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

Journal: World Journal of Surgical Oncology

Article Title: LncRNA NEAT1 mediates progression of oral squamous cell carcinoma via VEGF-A and Notch signaling pathway

doi: 10.1186/s12957-020-02028-x

Figure Lengend Snippet: LncRNA NEAT1 highly expressed in OSCC cell lines and promoted proliferation and EMT but inhibited apoptosis. a Expressions of lncRNA NEAT1 in HOEC, SCC-25, TSCC1, and CAL-27 cell lines were examined with RT-qPCR with normalizing to GAPDH and the statistical test was comparing each of the OSCC cell lines to the HOEC cell line, ** P < 0.05. b After suppression, levels of siNEAT1-1, siNEAT1-2, and siNC were checked by RT-qPCR with normalizing to GAPDH and the statistical test was comparing siNEAT1-1 and siNEAT1-2 to siNC, ** P < 0.05. c CCK-8 was assessed to measure cell viabilities in TSCC1 cell line with siNC and siNEAT1-2 and the statistical test was comparing siNEAT1-2 to siNC, ** P < 0.05. d Apoptosis rate of cells in TSCC1 cell line with transfected siNEAT1-2 and siNC were validated using flow cytometry and the statistical test was comparing siNEAT1-2 to siNC, ** P < 0.05. e Expressions of E-cadherin, N-cadherin, Vimentin, and Snail were checked with RT-qPCR with normalizing to GAPDH and the statistical test was comparing E-cadherin, N-cadherin, Vimentin, and Snail in siNEAT1-2 group to E-cadherin, N-cadherin, Vimentin, and Snail in siNC group, ** P < 0.05. Each experiment was repeated three times

Article Snippet: The human oral epithelial cell line HOEC and human OSCC cell lines (SCC-25, TSCC1, and CAL-27) were all acquired from American Type Culture Collection (ATCC, USA).

Techniques: Quantitative RT-PCR, CCK-8 Assay, Transfection, Flow Cytometry

Journal: World Journal of Surgical Oncology

Article Title: LncRNA NEAT1 mediates progression of oral squamous cell carcinoma via VEGF-A and Notch signaling pathway

doi: 10.1186/s12957-020-02028-x

Figure Lengend Snippet: VEGF-A expressed higher in OSCC cell lines with accelerating proliferation and EMT. a RNA expressions of VEGF-A were measured in HOEC, SCC-25, TSCC1, and CAL-27 cell lines using RT-qPCR with normalizing to GAPDH and the statistical test was comparing each of the OSCC cell lines to the HOEC cell line, ** P < 0.05. b RT-qPCR was assessed to evaluated level of VEGF-A after inhibition normalized with GAPDH and the statistical test was comparing siVEGF-A to siNC, ** P < 0.05. c Cell viabilities were checked by CCK-8 in TSCC1 cell line with suppressed VEGF-A with the statistical test comparing siVEGF-A to siNC, ** P < 0.05. d RT-qPCR was performed to check RNA levels of E-cadherin, N-cadherin, Vimentin, and Snail with normalizing to GAPDH and the statistical test was comparing E-cadherin, N-cadherin, Vimentin, and Snail in siVEGF-A group to E-cadherin, N-cadherin, Vimentin, and Snail in siNC group, ** P < 0.05. Each experiment was repeated three times

Article Snippet: The human oral epithelial cell line HOEC and human OSCC cell lines (SCC-25, TSCC1, and CAL-27) were all acquired from American Type Culture Collection (ATCC, USA).

Techniques: Quantitative RT-PCR, Inhibition, CCK-8 Assay

Journal: World Journal of Surgical Oncology

Article Title: LncRNA NEAT1 mediates progression of oral squamous cell carcinoma via VEGF-A and Notch signaling pathway

doi: 10.1186/s12957-020-02028-x

Figure Lengend Snippet: LncRNA NEAT1 accelerated proliferation and EMT and repressed apoptosis of OSCC cells through activating Notch signaling pathway. a Expressions of Notch1, Notch2, and Jagged1 with overexpressed NEAT1-2 and VEGF-A were evaluated by RT-qPCR normalized with GAPDH and the statistical test was comparing oeNEAT1-2 and oeNEAT1-2+oeVEGF-A to oeNC, ** P < 0.05. b Cell viabilities were checked in TSCC1 cells with overexpressed NEAT-1, overexpressed VEGF-A and IMR-1 via CCK-8 with the statistical test was comparing oeNEAT1-2, oeNEAT1-2+oeVEGF-A, and oeNEAT1-2+oeVEGF-A+IMR-1 to oeNC, ** P < 0.05. c Flow cytometry was performed to measure apoptosis rate of TSCC1 cell line with overexpressed NEAT1-2 and VEGF-A and added IMR-1 with the statistical test was comparing oeNEAT1-2, oeNEAT1-2+oeVEGF-A, and oeNEAT1-2+oeVEGF-A+IMR-1 to oeNC, ** P < 0.05. d RNA levels of E-cadherin, N-cadherin, Vimentin, and Snail were detected with overexpressed NEAT1-2, overexpressed VEGF-A and IMR-1 using RT-qPCR with normalizing to GAPDH and the statistical test was comparing E-cadherin, N-cadherin, Vimentin, and Snail in oeNEAT1-2, oeNEAT1-2+oeVEGF-A, and oeNEAT1-2+oeVEGF-A+IMR-1 group to E-cadherin, N-cadherin, Vimentin, and Snail in oeNC group, ** P < 0.05. Each experiment was repeated three times

Article Snippet: The human oral epithelial cell line HOEC and human OSCC cell lines (SCC-25, TSCC1, and CAL-27) were all acquired from American Type Culture Collection (ATCC, USA).

Techniques: Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry

Journal:

Article Title: The coordinate regulation of the p53 and mTOR pathways in cells

doi: 10.1073/pnas.0502857102

Figure Lengend Snippet: mTOR inhibition by p53 in MEFs requires both TSC1 and TSC2. (A) TSC1-/- MEFs. (B) WTor TSC2-/- p53-/- MEFs, as indicated, were treated with 20 μM etoposide for the indicated amount of time. (C) TSC2-/- p53-/- MEFs were transiently transfected with either p53 expression vector or empty control vector as indicated. Twenty-four hours posttransfection, the cells were treated with 20 μM etoposide as indicated or left untreated for 8 h (lanes 3 and 4) or 12 h (lanes 1, 2, 5, and 6). Cells from A, B, and C were collected, and the levels of phosphorylated p70S6K (T389), p53, and actin were determined by Western blot analyses.

Article Snippet: Assay-on-demand for human tuberin ( TSC2 ) (catalog no. Hs00241068_s1), human PTEN (catalog no. Hs00829813_m1), human actin (catalog no. Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal:

Article Title: The coordinate regulation of the p53 and mTOR pathways in cells

doi: 10.1073/pnas.0502857102

Figure Lengend Snippet: The mRNA and protein levels of PTEN and TSC2 are up-regulated upon p53 activation in V138 cells. (A and C) V138 cells and the parental H1299 cells were cultured at 37°C for 24 h before being shifted to 32°C for the indicated amount of time. Cells were collected, and total RNA were prepared. The mRNA levels of TSC2 (A) and PTEN (C) were determined by real-time PCR and normalized against the mRNA levels of actin. (B and D) V138 cells were cultured at 37°C for 24 h before being shifted to 32°C for the indicated amount of time. Cells were collected, and the protein levels of TSC2 (B) and PTEN (D) were detected by Western blot analyses. The levels of protein expression were quantified by densitometry and normalized against those of actin.

Article Snippet: Assay-on-demand for human tuberin ( TSC2 ) (catalog no. Hs00241068_s1), human PTEN (catalog no. Hs00829813_m1), human actin (catalog no. Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Activation Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Oncology Letters

Article Title: Heavy-ion beam irradiation inhibits invasion of tongue squamous cell carcinoma Tca8113 cells

doi: 10.3892/ol.2019.10761

Figure Lengend Snippet: Cell invasion assay. (A) Invasive potential of Tca8113 cells treated with X-rays or carbon ions at different doses. ×100 magnification was used. (B) Quantification of invasion assay results (n=3). *P<0.05 vs. X-ray.

Article Snippet: The human TSCC cell line Tca8113 was obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Invasion Assay

Journal: Oncology Letters

Article Title: Heavy-ion beam irradiation inhibits invasion of tongue squamous cell carcinoma Tca8113 cells

doi: 10.3892/ol.2019.10761

Figure Lengend Snippet: Expression of vascular endothelial growth factor (green) in Tca8113 cells irradiated with X-rays at different doses. An indirect immunofluorescence assay was performed at 12, 24 or 48 h post-irradiation. ×1000 magnification was used.

Article Snippet: The human TSCC cell line Tca8113 was obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Irradiation, Immunofluorescence

Journal: Oncology Letters

Article Title: Heavy-ion beam irradiation inhibits invasion of tongue squamous cell carcinoma Tca8113 cells

doi: 10.3892/ol.2019.10761

Figure Lengend Snippet: Expression of vascular endothelial growth factor in Tca8113 cells (green) irradiated with carbon ions at different doses. An indirect immunofluorescence assay was performed at 12, 24 or 48 h post-irradiation. ×1000 magnification was used.

Article Snippet: The human TSCC cell line Tca8113 was obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Irradiation, Immunofluorescence

Journal: Oncology Letters

Article Title: Heavy-ion beam irradiation inhibits invasion of tongue squamous cell carcinoma Tca8113 cells

doi: 10.3892/ol.2019.10761

Figure Lengend Snippet: Quantification of vascular endothelial growth factor in Tca8113 cells irradiated with (A) X-rays and (B) carbon ions. *P<0.05 vs. control group.

Article Snippet: The human TSCC cell line Tca8113 was obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Irradiation, Control