Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum aggregates at the invasive margin of OSCC and correlates with tumor invasion. (A) F. nucleatum abundance for adjacent normal tissues (right) and OSCC cancerous tissues (left) is plotted. A total of 70 matched OSCC tissue pairs were tested. Each symbol represents data from one sample (OSCC cancerous tissue and adjacent normal tissue) (one-way-ANOVA), P = 0.0006. (B–D) F. nucleatum abundance for OSCC tissues with or without cervical lymph node metastasis (B), at high, middle and low pathological stages (C), as well as with flange, spike and skipping invasive phenotypes (D). (one-way-ANOVA), (B), P = 0.0231; (C), ANOVA P = 5.0E-16; (D), ANOVA P = 7.9E-05. (E) Representative images of H&E staining for tissue structure (up) and F. nucleatum spatial distribution by FISH staining (down) from clinical samples of OSCC lesions with flange, spike and skipping invasive phenotypes, respectively. Black and white curves indicate the borders between tumor tissues and adjacent normal tissues. (F) Representative data of xenograft tumors in C3H mice bearing SCC-7 cells in different groups. Local injection of F. nucleatum was applied to establish the F. nucleatum -associated tumor model. (G and H) Statistical analysis of mouse tumor weights (I) and volumes (J) in different groups. n = 5/group (two-tailed t test), Weight, P = 3.5E-05; Volume, P = 0.0052. (I) Representative FISH images of clinical OSCC samples using F. nucleatum -specific 16S rDNA-directed probe (left) and fluorescent images stained with 488-anti-SOX2, 488-anti-N-cad and 488-anti-ZEB1 (right) for measurement of tumor invasive levels by successive sections. (J) Representative fluorescent images of xenograft tumor samples with or without F. nucleatum infection stained with SOX2 (green), N-cad (green) and DAPI (blue).

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Staining, Injection, Two Tailed Test, Infection

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: Colonized F. nucleatum drives M2-like tumor-associated macrophages formation. (A and B) Representative fluorescent images of F. nucleatum spatial distribution, total macrophages and TAMs enrichment status in successive sections from clinical OSCC samples. Cy3-labeled F. nucleatum -specific probe was used for the detection of F. nucleatum . 488-anti-CD68 and 488-anti-CD206 were applied to visualize total macrophages and TAMs, respectively. A total of 40 paired successive sections on OSCC primary locus were tested to explore the relationship between F. nucleatum colonization and macrophage accumulation. (C) The Pearson linear correlation analysis was conducted based on F. nucleatum counts, total macrophages and TAMs in OSCC. (D) Representative fluorescent images of xenograft tumor samples (left) or normal oral epithelial samples (right) from C3H mice with or without F. nucleatum colonization stained with Cy3-anti-CD206 for identification of TAMs. (E) Representative data of xenograft tumors in C3H mice bearing SCC-7 cells in different groups. Local injection of F. nucleatum was used for in vivo F. nucleatum -associated tumor model. Injection of clodronate liposomes was applied for the eradication of local macrophages. (F and G) Statistical analysis of mouse tumor weights (I) and volumes (J) in different groups. n = 5/group (two-tailed t test), Weight, P = 0.0001; Volume, P = 4.9E-07. (H) The fluorescent assay showed a significant reversal of F. nucleatum -induced EMT markers (SOX2 and ZEB1) upregulation by eradicating local macrophages using clodronate liposomes. (I) schematic graph of indirect co-culture system including two crucial steps: extraction of oral tumor/epithelial cell culturing supernatant with or without F. nucleatum infection, and stimulation of M0 macrophages by extracted cell supernatant. (J and K) Assessment of macrophage polarization status in the indirect co-culture system mentioned above by immunofluorescent assay for CD86+ (green) M1-like macrophages and CD206+ (red) M2-like macrophages derived from M0 macrophages (J) as well as measuring mRNA expression of M2 markers ( CCL2 and IL12 ) in macrophages (K) indirectly co-cultured with oral tumor/epithelial cells with or without F. nucleatum infection (two-tailed t test), CCL2 , P = 0.0165; IL12 , P = 6.5E-05.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Labeling, Staining, Injection, In Vivo, Two Tailed Test, Fluorescence, Co-Culture Assay, Cell Culture, Infection, Derivative Assay, Expressing

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum -induced lactate production of OSCC cells is required for M2-like tumor-associated macrophages formation. (A) Representative Giemsa staining images of OSCC invasive margins. The red square indicates the acidic border between tumor tissue and adjacent normal tissue, and the orange square indicates the non-acidic central regions. (B) Volcano plot for metabolite changes in filtered cell culturing supernatant with or without F. nucleatum infection. The red and green symbols indicate significantly upregulated and downregulated metabolites between the two groups. (C) Metabolic classification analysis of the top 5 metabolic terms based on differential metabolites in different groups (ranked by the number of enriched metabolites). (D) Pathway enrichment analysis of the top 5 metabolic pathways based on differential metabolites between the above two groups. (E) Alteration of intermediate metabolites of glycolysis in CAL27 culturing supernatant with or without infection of F. nucleatum (two-tailed t test), G6P, P = 0.0006; F-1,6-bisP, P = 1.2E-06; G3P, P = 0.0003; PEP, P = 0.0022; Pyruvate, P = 0.0004; Lactate, P = 0.0037. (F) Infection of F. nucleatum significantly enhanced the production of lactate, pyruvate, glucose consumption, and production of ATP in CAL27 cells. (two-tailed t test), Lactate, P = 0.0001; Pyruvate, P = 2.4E-06; Glucose consumption, P = 2.3E-05; ATP production, P = 0.0018. (G) Schematic graph of indirect co-culture system includes two crucial steps: extraction of CAL27 cell culturing supernatant with or without F. nucleatum infection, and stimulation of M0 macrophages by extracted cell supernatant with or without addition of 7ACC2 (10 μM) for inhibition of lactate adsorption. (H and I) Assessment of macrophage polarization status by measuring mRNA expression of M2 markers ( CCL2 and IL12 ) (I) as well as immunofluorescent assay of CD206 (J) in CAL27 cells treated with DMSO, F. nucleatum suspension or 7ACC2. (one-way-ANOVA), CCL2 , ANOVA P = 0.0048; IL12 , ANOVA P = 5.3E-05. (J) Schematic graph of indirect co-culture system including two crucial steps: extraction of oral tumor cell culturing supernatant with or without F. nucleatum infection plus 2-DG (20 mM) for inhibition of glycolysis, and stimulation of M0 macrophages by extracted cell supernatant. (K and L) Assessment of macrophage polarization status by measuring mRNA expression of M2 markers ( IL10 and IL12 ) (I) as well as immunofluorescent assay of CD206 (J) in CAL27 cells treated with DMSO, F. nucleatum suspension or 2-DG. (one-way-ANOVA), CCL2 , ANOVA P = 0.0141; IL12 , ANOVA P = 1.7E-06. (M) Schematic graph depicting indirect co-culture system with F. nucleatum -mediated TAM failed to promote tumor invasive behavior by inhibiting lactate adsorption of macrophages using 7ACC2. (N and O) Fluorescence assay of CAL27 cells for expression levels of ZEB1 and VIMENTIN (left) and Transwell assay depicting OSCC invasive potential under the above conditions. (one-way-ANOVA), ANOVA P = 0.0005.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Staining, Cell Culture, Infection, Two Tailed Test, Co-Culture Assay, Inhibition, Adsorption, Expressing, Fluorescence, Transwell Assay

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum -induced accumulation of GLUT1 increases lactate production of OSCC cells. (A) Western blot analysis of glycolysis-related rate-limiting components (HK2, PFKP, PKM2, ENO1, LDHA, GLUT1) and GAPDH (for loading controls) in CAL27 cells co-cultured with or without F. nucleatum . All experiments were performed repeatedly three times. (two-tailed t test), PKM2, P = 0.048; LDHA, P = 0.0419; GLUT1, P = 0.0148. (B) Representative fluorescent images of successive sections from OSCC tissues for localization of F. nucleatum (red) and GLUT1 (green). (C) Representative images of GLUT1 (green) expression pattern in OSCC. (D) Clinical OSCC sections stained with 488-anti-KRT19 (green) and Cy3-anti-GLUT1 (red). High expression levels of GLUT1 could be detected mainly in KRT19+ tumor cells. (E) Representative images of xenograft tumor sections for measurement of GLUT1 expression (green) after injection of F. nucleatum suspension or PBS vehicle. (F and G) Glucose uptake and lactate production in CAL27 cells treated with F. nucleatum infection, BAY-876 (50 nM) or GLUT1 knockdown operations. (one-way-ANOVA), (F), Glucose, ANOVA P = 1.7E-06; Lactate, ANOVA P = 0.0142. (G), Glucose, ANOVA P = 0.0025; Lactate, ANOVA P = 0.0049. (H and I) Evaluation of macrophage polarization status by confocal fluorescence assay for CD206 (H) and mRNA expression levels of CCL2 and IL-12 (I). Macrophages were indirectly co-cultured with CAL27 cells treated by PBS, F. nucleatum infection or BAY-876. (one-way-ANOVA), CCL2 , ANOVA P = 0.0001; IL12 , ANOVA P = 0.0216.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Western Blot, Cell Culture, Two Tailed Test, Expressing, Staining, Injection, Infection, Fluorescence

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum promotes GLUT1 localization on the cell surface through autophagy-dependent TBC1D5 decrease. (A) Representative confocal images of GLUT1 (green) and DIL-labelled cellular membrane (red) in CAL27 cells with or without F. nucleatum co-culture. (B) Western blot analysis of GLUT1 in the cytoplasm and cellular membrane of CAL27 cells with or without F. nucleatum infection. GAPDH was applied as the loading control for cytoplasm protein, and ATP1A was used as the loading control for membrane protein (two-tailed t test), P = 0.0002. (C) Western blot analysis of GLUT1 in CAL27 cells with or without F. nucleatum infection after pretreatment of CHX for 6 h at the proper concentration of 50 μg/mL. GAPDH was applied as the loading control. (one-way-ANOVA), ANOVA P = 7.4E-09. (D) Fluorescence assay for colocalization of GLUT1 (green) and LAMP1 (red) in CAL27 cells with or without F. nucleatum infection. (E) Western blot analysis of TBC1D5 and GLUT1 in CAL27 and CAL27/TBC1D5 treated by F. nucleatum infection or not. (one-way-ANOVA), TBC1D5, ANOVA P = 1.2E-05; GLUT1, ANOVA P = 0.0076. (F) The functional enrichment analysis of differentially expressed genes between CAL27 cells with or without F. nucleatum infection. (G) Confocal images of BECLIN1 (green) and DIL-labelled cellular membrane (red) in CAL27 cells with or without F. nucleatum infection. (H) Indicated proteins were detected by Western blot in CAL27 cells. The cells were co-cultured with F. nucleatum or treated by 3-MA (12 mM) for inhibition of autophagy. (one-way-ANOVA), TBC1D5, ANOVA P = 0.0040; GLUT1, ANOVA P = 2.3E-05; BECLIN1, ANOVA P = 1.1E-05; ATG5, ANOVA P = 0.0008. (I) Measurement of glucose uptake in CAL27 cells treated by PBS, F. nucleatum suspension or 3-MA. (one-way-ANOVA), ANOVA P = 0.0005.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Co-Culture Assay, Western Blot, Infection, Two Tailed Test, Concentration Assay, Fluorescence, Functional Assay, Cell Culture, Inhibition

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum binds to GalNAc on OSCC cell surface to induce cell autophagy. (A) Fluorescence assay of KRT19 (red) and GalNAc (green) in clinical OSCC sections. (B) Representative fluorescent images of F. nucleatum (red) and FITC-PNA (green) in clinical OSCC sections. (C) Confocal images of SYTO9-labelled F. nucleatum (green) in CAL27 cells treated by PBS or PNA solutions at the proper concentration of 80 μg/mL. (D and E) Western blot analysis of indicated proteins in CAL27 cells treated with PBS, F. nucleatum suspension or PNA solution (one-way-ANOVA), ATG5, ANOVA P = 0.0004; BECLIN1, ANOVA P = 0.0016. (F) Glucose consumption (left) and lactate production (right) in CAL27 cells under F. nucleatum infection with or without PNA treatment. (one-way-ANOVA), Glucose, ANOVA P = 1.9E-07; Lactate, ANOVA P = 3.8E-08. (G and H) Confocal images of GLUT1 (green), LAMP1 (red), DIL-labelled cellular membrane (red) and cellular nuclei (blue) in CAL27 cells under F. nucleatum infection for 12 h and 24 h pretreated by PNA solution for localization of cellular GLUT1. (I) Western blot analysis of key components in AKT/mTOR pathway in CAL27 cells treated by F. nucleatum suspension or PNA solution. (one-way-ANOVA), P-AKT, ANOVA P = 2.3E-06; p-mTOR, ANOVA P = 0.0003. (J) Western blot analysis of GLUT1, BECLIN1 and ATG5 in CAL27 cells under F. nucleatum , SC79 or 3BDO treatment. GAPDH was used as the loading control. GLUT1, (one-way-ANOVA), ANOVA P = 0.0002; BECLIN1, ANOVA P = 2.0E-09; ATG5, ANOVA P = 0.0001.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Fluorescence, Concentration Assay, Western Blot, Infection

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: Double targeting at GalNAc and GLUT1 inhibits OSCC progression. (A) Workflow diagram for translational medical research using BAY-876 and PNA for treatment of OSCC. (B) Representative images of xenograft tumors in C3H mice bearing SCC-7 cells in different groups. Local injection of F. nucleatum was used for in vivo F. nucleatum -associated tumor model, and injection of PNA, BAY-876 and a combination of them were applied as a different therapeutic strategy. (C and D) Statistical analysis of mouse tumor weights (C) and volumes (D) in different groups. n = 5/group. (one-way-ANOVA), Weight, ANOVA P = 1.7E-06; Volume, ANOVA P = 2.3E-08. (E) Representative images of CD68+ CD206+ M2-like TAMs (above) and CD86+ M1-like anti-tumor macrophages (below) in xenograft tumor samples from different groups. (F) Representative images of SOX2 (above) and ZEB1 (below) in xenograft tumor samples from different groups.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Injection, In Vivo

Journal: Cell genomics

Article Title: A cis -regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation

doi: 10.1016/j.xgen.2022.100191

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CAL27 human squamous cell carcinoma cell line was obtained from ATCC (CRL-2095) and grown in DMEM F:12 (ThermoFisher, 11995–065) supplemented with 10% FBS.

Techniques: Recombinant, SYBR Green Assay, Staining, HiChIP, Software

Journal: Cell genomics

Article Title: A cis -regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation

doi: 10.1016/j.xgen.2022.100191

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: CAL27 , ATCC , Cat#CRL-2095; RRID:CVCL_1107.

Techniques: Recombinant, SYBR Green Assay, Staining, HiChIP, Software

Journal: Communications Biology

Article Title: Role of miR-944/MMP10/AXL - axis in lymph node metastasis in tongue cancer

doi: 10.1038/s42003-023-04437-6

Figure Lengend Snippet: a qRT-PCR and immunoblot of MMP10 and GAPDH in AW13516 cells stably overexpressing empty vector or MMP10 . Numbers on the blot indicate intensity ratio of MMP10 expression with respect to the vector control lane. b , f , j Wound-healing assay of AW13516 cells stably overexpressing empty vector or MMP10 ( b ), AW8507 cells ( f ) and CAL27 cells ( j ) with non-targeting shRNA (sh-NT) or stable MMP10 knockdown (3 shRNAs – sh1 or sh-2 or sh-3). Representative images of wound-healing assay at 0 h and 21 h are presented along with the bar plots indicating the percentage of wound closure. c , g , k Boyden chamber matrigel invasion assay of AW13516 cells stably overexpressing empty vector or MMP10 ( c ), AW8507 cells ( g ) and CAL27 cells ( k ) with sh-NT or stable MMP10 knockdown with sh1 or sh-2 or sh-3. Representative images of crystal violet stained Boyden chamber along with the bar plots depict the percent cell invasion (scale bar = 400 µm). d , h qRT-PCR and immunoblot of MMP10 and GAPDH in AW8507 ( d ) and CAL27 (h) cells with sh-NT or stable MMP10 knockdown. Numbers on the blot indicate intensity ratio of MMP10 expression with respect to the sh-NT control lane. e , i Cell proliferation assay of AW8507 ( e ) and CAL27 cells ( i ) with sh-NT or stable MMP10 knockdown with sh-1 or sh-2 or sh-3. Scatter plots indicate the number of live cells on the mentioned day. Data are shown as means ± SD. p -values are from Student’s unpaired t -test and denoted as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data shown are representative of n = 3 independent experiments.

Article Snippet: AW13516 and AW8507 tongue cancer cell lines were obtained from Tata Memorial Hospital while CAL27 cells were procured from ATCC.

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Plasmid Preparation, Expressing, Wound Healing Assay, shRNA, Invasion Assay, Staining, Proliferation Assay

Journal: Cancers

Article Title: The Effect of Cannabis Plant Extracts on Head and Neck Squamous Cell Carcinoma and the Quest for Cannabis-Based Personalized Therapy

doi: 10.3390/cancers15020497

Figure Lengend Snippet: Differential effect of different Cannabis extracts on the viability of HNSCC cells. ( A ) Cell viability of HNSCC cell lines Scc4, Scc9, Scc25, and Cal27 following treatment with 24 cannabis extracts at 6 µg/mL concentration for 24 h. Cell viability was evaluated by MTT, and data are reported as mean ± SE (n = 3) of % dead cells compared to DMSO control, % Dead cells = 100 × ( Control − Sample ) Control . Differences were statistically analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test (** p < 0.001, **** p < 0.0001). ( B ) Dose-response curve of cell lines Scc4, Scc9, Scc25, and Cal2, following 24 h of treatment with CAN296 (decarboxylated CBD-type) extract. Data are reported as mean ± SE (n = 3) of % Dead cells compared to DMSO control. Statistically analyzed with two-way ANOVA followed by Tukey’s multiple comparisons (* p < 0.05).

Article Snippet: Four well-characterized human adherent epithelial cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA): Scc4 (ATCC ® CRL1624 TM ), Scc9 (ATCC ® CRL1629 TM ), Scc25 (ATCC ® CRL1628 TM ), and Cal27 (ATCC ® CRL2095 TM ).

Techniques: Concentration Assay

Journal: Cancers

Article Title: The Effect of Cannabis Plant Extracts on Head and Neck Squamous Cell Carcinoma and the Quest for Cannabis-Based Personalized Therapy

doi: 10.3390/cancers15020497

Figure Lengend Snippet: Apoptotic effect of Cannabis extracts on HNSCC. Extract CAN296 (2, 4, 8 µg/mL) was applied on Scc25 or Cal27 cells for 12 h, with DMSO as the control. ( A ) Apoptosis (early and late) was assessed by APC Annexin-V/PI staining with flow cytometry. ( B ) The percent of apoptotic cells was calculated as % of positive Annexin-V APC cells out of the total cells counted (events = 10,000) and presented as mean ± SE (n = 3). Statistically analyzed with two-way ANOVA followed by Tukey’s multiple comparisons test, and asterisks indicate significant differences compared to the control (**** p < 0.0001). ( C ) Cells were lysed and resolved on 15% SDS-PAGE, followed by western blotting with anti-Cleaved Caspase 3, c.PARP and GAPDH as the loading control. ( D ) Scc25 cells were treated with 0.5–8 µg/mL CAN296 extract for either 24 or 72 h, and cell proliferation was assessed according to WST-1 assay. The percent of vital cells relative to DMSO (control) is presented as mean ± SE (n = 3). Asterisks indicate statistical significance between 24 h and 72 h treatment (** p < 0.0005, **** p < 0.0001; two-way ANOVA with Sidak’s post-hoc multiple comparison test).

Article Snippet: Four well-characterized human adherent epithelial cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA): Scc4 (ATCC ® CRL1624 TM ), Scc9 (ATCC ® CRL1629 TM ), Scc25 (ATCC ® CRL1628 TM ), and Cal27 (ATCC ® CRL2095 TM ).

Techniques: Staining, Flow Cytometry, SDS Page, Western Blot, WST-1 Assay

Journal: Frontiers in Oncology

Article Title: Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma

doi: 10.3389/fonc.2022.1085917

Figure Lengend Snippet: STIP1 is overexpressed in OSCC-derived cell lines. (A) Total RNA from the normal human gingival keratinocyte cell line (HGK) and 6 human OSCC cell lines (SCC4, SCC9, SCC15, SCC25, HSC3 and CAL27) were converted in cDNA and subjected to qPCR. The STIP1 mRNA levels were significantly higher in OSCC cells lines than in HGK, with exception of CAL27. (B) The overexpression of STIP1 was confirmed with western blot applying specific antibodies against STIP1 and the housekeeping control β-actin. (C) Immunofluorescence analysis revealed an intense staining for STIP1 in OSCC cells, which was mainly found in the perinuclear area. STIP1 knockdown efficiency in HSC3 and SCC9 cells after transduced with lentivirus expressing shRNA sequences against STIP1 (clones #1 and #2) and control as outlined in the methods. A marked reduction in both mRNA (D) and protein (E) levels when compared with control cells was observed. The values represent the average ± SD of three separate experiments. In figures (B, C, E) a representative image is shown. *p<0.01, **p<0.0001.

Article Snippet: The human OSCC cell lines SCC4 (CRL-1624), SCC9 (CRL-1629), SCC15 (CRL-1623), SCC25 (CRL-1628), CAL27 (CRL-2095) (ATCC, Manassas, VA, USA) and HSC3 (JCRB 0623; Osaka National Institute of Health Sciences, Japan) were cultured in DMEM/F12 medium (1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS), 400 ng/ml hydrocortisone (Sigma-Aldrich, USA) and a mixture of antibiotic-antimycotic (Invitrogen, USA).

Techniques: Derivative Assay, Over Expression, Western Blot, Immunofluorescence, Staining, Transduction, Expressing, shRNA, Clone Assay

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: Chrysophanol reduced cell viability and proliferation in CAL-27 and Ca9-22 cells. ( A , B ) Both cells were incubated with several concentrations of chrysophanol for 24 to 72 h (0–300 μM), and then the viability of cells was measured using the MTT assay. ( C – F )) A colony-formation assay was performed to examine cell proliferation. Cells were treated with chrysophanol for seven days and stained with a 1% crystal violet solution. The number of colonies was converted into a percentage and shown in a histogram. The results are exhibited as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: Incubation, MTT Assay, Colony Assay, Staining

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: Chrysophanol induced apoptosis via caspase activations in CAL-27 and Ca9-22 cells. Both cells were incubated with chrysophanol (0, 100, and 150 μM) for 24 h. ( A , B ). The change in nuclear morphology was investigated by Hoechst agents. Then, nuclear condensation was converted into proportions to control the process and is shown in a histogram. ( C , D ) After the treatment of 200 μM chrysophanol, the mitochondrial membrane potential (ΔΨm) was measured by JC-1 solutions. ( E ) Western blot analysis was used to identify the expression level of proteins which affect cell apoptosis, such as Bax, Bcl-2, caspase-3, caspase-7, and PARP. β-actin was used to present a loading control. ( F , G ) The relative protein, cleaved PARP/PARP, and Bax/Bcl-2 density were measured; and the fold change was calculated. Data are expressed as mean ± SD (* p < 0.05, ** p < 0.01).

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: Incubation, Western Blot, Expressing

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: Chrysophanol-induced autophagy in CAL-27 and Ca9-22 cells. ( A , B ) The cells were treated with 100 μM chrysophanol. Then, acidic vesicular organelles’ (AVOs) formation was observed by AO staining, and MDC staining was used to detect the autophagic vacuole accumulation. ( C , D ) The expression levels of autophagy-related protein ATG5, beclin-1, LC3B-I/LC3B-II, and p62 were examined using a Western blot analysis, and the density of protein LC3B-II/LC3B-I ratios is shown in the graph. β-actin was used to present a loading control. ( E – G ) Autophagy-related mRNA expression levels, such as those of ATG5, beclin-1, p62/SQSTM1, and MAP1LC3B, were identified using a real-time PCR. GAPDH was used to present a loading control. Data are expressed as mean ± SD (* p < 0.05).

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: Staining, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: It was found that 3-MA inhibited autophagy and cooperated with apoptosis, which was occasioned by chrysophanol in CAL-27 and Ca9-22 cells. ( A , B ) Cell viability was executed by an MTT assay after chrysophanol treatments with or without 3-MAfor 24 h. ( C , D ) The protein expression levels of autophagy-associated proteins (ATG5, beclin-1, and LC3B) and apoptosis-associated proteins (Bax, Bcl-2, Caspase -3, and PARP) were detected by using a Western blot analysis. β-actin was used to present a loading control. ( E – G ) Bax/Bcl-2, cleaved caspase -3/caspase -3, and cleaved PARP/PARP ratios were calculated by using the Western blot band density. ( H , I ) Chrysophanol (0, 150 μM) was treated with both cells in the presence or absence 3-MA, and then the revelation of cytochrome c was visualized by immunofluorescence analyses. The results are exhibited as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: MTT Assay, Expressing, Western Blot, Immunofluorescence

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: Chrysophanol enhanced the Akt/mTOR pathway in CAL-27 and Ca9-22 cells. ( A , B ) Chrysophanol was applied at 0 to 200 μM for 24 h in both OSCC cells. The expression of levels of Akt, p-Akt, mTOR, and p-mTOR were obtained using Western blot analysis and the p-Akt/Akt Western blot band density ratio is shown in the histogram. β-actin was used to present loading controls. Data are expressed as mean ± SD (* p < 0.05).

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: Expressing, Western Blot

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: Inhibition of the PI3K/Akt/mTOR pathway promoted chrysophanol-induced apoptosis in CAL-27 and Ca9-22 cells. Both cells were pre-treated with 20 μM LY294002 for 2 h and then treated with chrysophanol for 24 h. ( A , B ) The expression levels of Akt, p-Akt, mTOR, and p-mTOR were detected using Western blot analyses, and the relative protein p-Akt/Akt ratio was calculated by a Western blot band. ( C – G ) The protein expression levels of autophagy-related proteins (beclin-1, p62, and LC3B) and apoptosis-related proteins (Bax, Bcl-2, caspase -3, and PARP) were evaluated by Western blot analyses, and then the ratios of apoptosis-regulated proteins are indicated in the histogram. ( H , I ) Making observations for JC-1, cells were treated with 150 μM chrysophanol in the presence or absence of LY294002. Data are expressed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: Inhibition, Expressing, Western Blot

Journal: Medicina

Article Title: Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

doi: 10.3390/medicina59010042

Figure Lengend Snippet: Graphical illustration of apoptotic and autophagic effects induced by chrysophanol in human OSCC, CAL-27 and Ca9-22 cells. Chrysophanol activates autophagy and in parallel inhibits apoptosis activity. 3-MA, an autophagy inhibitor, inhibits autophagy and cooperates with apoptosis, which is occasioned by chrysophanol. Inhibition of the PI3K/Akt/mTOR signaling pathway with the PI3K inhibitor LY294002 enhances chrysophanol-induced apoptosis and reduces autophagy. Therefore, chrysophanol-induced autophagy acts as a cell protection mechanism and interferes with the apoptosis pathway of OSCC cells.

Article Snippet: CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA).

Techniques: Activity Assay, Inhibition