Journal: Cancers

Article Title: Drug Repurposing Applications to Overcome Male Predominance via Targeting G2/M Checkpoint in Human Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers14235854

Figure Lengend Snippet: Male ESCC cell lines were more sensitive to decitabine and MK1775 than females. ( a ) Workflow of drug repurposing by using the gene expression data of patients over 60 years old. ( b ) Ten drugs were selected from the drug repurposing results. ( c , d ), Growth curves of KYSE150, KYSE510, KYSE30, and KYSE450 cells were measured by IncuCyte S3 for 72 h. KYSE30 and KYSE450 cells were derived from male patients, while KYSE150 and KYSE510 cells were derived from female patients. Cells were treated with decitabine (10 μM) or MK1775 (200 nM). ( e – g ), Representative image, tumor weights, and tumor volumes of xenografts derived from KYSE30 cells (male), KYSE150 cells (female), and KYSE450 (male) that were treated with decitabine (1.0 mg/kg, i.p.) or MK1775 (60 mg/kg, p.o.). The data shown are the mean ± SD; n = 6 mice per group in KYSE30 and KYSE150; n = 5 mice per group in 450. For tumor weights, data were analyzed using two-tailed t-tests; for tumor volumes, data were analyzed using two-way ANOVA with Bonferroni correction. (* p < 0.05, ** p < 0.01, and *** p < 0.001; ns = not significant).

Article Snippet: A total of 1 × 10 6 KYSE150 cells were subcutaneously injected into 6-week-old female Balb/c nude mice (GemPharmatech Co., Ltd., Nanjing, China).

Techniques: Gene Expression, Derivative Assay, Two Tailed Test

Journal: Cancers

Article Title: Drug Repurposing Applications to Overcome Male Predominance via Targeting G2/M Checkpoint in Human Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers14235854

Figure Lengend Snippet: Validation RNA-seq proves that MK1775 and decitabine showed a sex-biased treatment response by targeting the G2/M checkpoint. ( a ) Drug-gene network between decitabine and MK1775 and their target genes was assessed in STICH. Gene functional ontology analysis in the ( b ) decitabine KYSE30 cell line, ( c ) MK1775 KYSE30 cell line, and ( d ) MK1775 KYSE150 cell line.

Article Snippet: A total of 1 × 10 6 KYSE150 cells were subcutaneously injected into 6-week-old female Balb/c nude mice (GemPharmatech Co., Ltd., Nanjing, China).

Techniques: Biomarker Discovery, RNA Sequencing, Functional Assay

Journal: American Journal of Cancer Research

Article Title: Hypoxia-induced lncRNA CASC9 enhances glycolysis and the epithelial-mesenchymal transition of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling

doi:

Figure Lengend Snippet: CASC9 enhances the glycolysis of pancreatic cancer cells. A. The relative expression of CASC9 was compared between different pancreatic cancer cell lines (BxPC-3, MiaPaCa-2, PANC-1, and SW1990) and a normal pancreatic duct cell line (HPDE6-C7) by using qRT-PCR. SW1990 and PANC-1 cell lines were transfected with Si-CASC9 or Si-NC. B. The relative mRNA level of CASC9 was detected by qRT-PCR. C. The ability of relative glucose uptake was measured by the glucose uptake assay. D. The relative production of lactate in each culture medium was determined by the lactate release assay. E. Western blot analysis was used to detect the protein levels of the key glycolytic enzymes HK2, GLUT4, and LDHA. Then, two pancreatic cancer cell lines were transfected with the pcDNA-CASC9 or pcDNA-NC plasmid. F. The mRNA level of CASC9 was determined by qRT-PCR. G and H. The relative abilities of glucose uptake and lactate production were measured by glucose uptake and lactate release assays, respectively. I. The protein levels of HK2, GLUT4, and LDHA were detected by western blot analysis. The results shown are from three independent experiments. The mRNA value was normalized to GAPDH. The lactate level was normalized to total protein of each sample. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After treatment, approximately 4 × 10 6 SW1990 cells (suspended in 0.2 mL of PBS) were injected into the lateral tail vein of athymic mice (7 to 8 weeks old; HFK Bioscience Co.; n = 5 per group).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Release Assay, Western Blot, Plasmid Preparation

Journal: American Journal of Cancer Research

Article Title: Hypoxia-induced lncRNA CASC9 enhances glycolysis and the epithelial-mesenchymal transition of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling

doi:

Figure Lengend Snippet: CASC9 enhances the EMT phenotype of pancreatic cancer cells. SW1990 and PANC-1 cells were transfected with Si-CASC9 or Si-NC and then subjected to the Transwell migration and invasion assays. A. The relative protein levels of Snail, Vimentin, N-Cadherin, and E-Cadherin were measured by western blot analysis. B. Representative images of migrated and invaded cells are shown on the membrane of the lower chamber. C. The relative abilities of migration and invasion are presented by quantification of stained cells. D. Two cell lines were transfected with the pcDNA-CASC9 or pcDNA-NC plasmid, and western blot analysis was used to detect the expression of Snail, Vimentin, N-Cadherin, and E-Cadherin. E and F. After transfection with pcDNA-CASC9 or pcDNA-NC, the relative migratory and invasive abilities in the two cell lines were determined by Transwell migration and invasion assays, respectively. The graphs presented are from three independent assays. Scale bar, 100 μm. **, P < 0.01.

Article Snippet: After treatment, approximately 4 × 10 6 SW1990 cells (suspended in 0.2 mL of PBS) were injected into the lateral tail vein of athymic mice (7 to 8 weeks old; HFK Bioscience Co.; n = 5 per group).

Techniques: Transfection, Migration, Western Blot, Membrane, Staining, Plasmid Preparation, Expressing

Journal: American Journal of Cancer Research

Article Title: Hypoxia-induced lncRNA CASC9 enhances glycolysis and the epithelial-mesenchymal transition of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling

doi:

Figure Lengend Snippet: CASC9 inhibition suppresses the tumorigenicity and metastasis of pancreatic cancer in vivo. LV-Si-CASC9- and LV-Si-NC-transfected SW1990 cells were implanted in the right flank of nude mice subcutaneously. After approximately 24 days, the mice were euthanized, and the tumor was completely removed. A. Representative photographs of mice and tumors are shown. B. The volume of the tumor was measured every 4 days, and the growth curve was delineated accordingly. C. The tumor weight was measured in each group. Then, the tumor tissue sections of xenografts in different groups were used for further analysis. D-F. The expression of the apoptotic marker TUNEL was stained using an immunofluorescence assay; the staining of Ki-67, HK2, GLUT4, LDHA, Snail, Vimentin, N-Cadherin, and E-Cadherin was analyzed by immunohistochemistry (scale bar, 50 μm). After being transfected with LV-Si-CASC9 or LV-Si-NC, SW1990 cells were injected into the lateral tail vein of mice. G. Representative photographs of resected lungs in each group are revealed. H. Representative H&E staining images of lungs are shown (scale bar, 200 μm). The metastatic nodules are indicated by arrows. I and J. The number of mice with lung metastasis and metastatic nodules were calculated in the two groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After treatment, approximately 4 × 10 6 SW1990 cells (suspended in 0.2 mL of PBS) were injected into the lateral tail vein of athymic mice (7 to 8 weeks old; HFK Bioscience Co.; n = 5 per group).

Techniques: Inhibition, In Vivo, Transfection, Expressing, Marker, TUNEL Assay, Staining, Immunofluorescence, Immunohistochemistry, Injection

Journal: American Journal of Cancer Research

Article Title: Hypoxia-induced lncRNA CASC9 enhances glycolysis and the epithelial-mesenchymal transition of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling

doi:

Figure Lengend Snippet: AKT activation participates in the enhancement of glycolytic flux and EMT partially mediated by CASC9-induced HIF-1α activation. SW1990 and PANC-1 cells were transfected with pcDNA-CASC9 or pcDNA-NC, followed by treatment with 20 μM LY294002 for 24 h. A. Western blot analysis was used to detect the protein levels of HK2, GLUT4, LDHA, Snail, Vimentin, N-Cadherin, E-Cadherin, HIF-1α, and p-AKT. B and C. The relative abilities of glucose uptake and lactate release were determined by the glucose uptake and lactate release assays, respectively. D-F. The relative migratory and invasive abilities of the two cell lines were measured by the Transwell migration and invasion assays, respectively. The graphs shown are from three independent assays. The lactate value was normalized to total protein of each sample. Scale bar, 100 μm. *, P < 0.05; **, P < 0.01.

Article Snippet: After treatment, approximately 4 × 10 6 SW1990 cells (suspended in 0.2 mL of PBS) were injected into the lateral tail vein of athymic mice (7 to 8 weeks old; HFK Bioscience Co.; n = 5 per group).

Techniques: Activation Assay, Transfection, Western Blot, Migration

Journal: American Journal of Cancer Research

Article Title: Hypoxia-induced lncRNA CASC9 enhances glycolysis and the epithelial-mesenchymal transition of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling

doi:

Figure Lengend Snippet: Hypoxia potentiates HIF-1α-regulated CASC9 transcriptionally. (A) SW1990 and PANC-1 cells were incubated under the hypoxic (1%) condition for different durations, and the mRNA level of CASC9 was measured by qRT-PCR. (B) Two cell lines were treated with 100 μM CoCl2 for 24 h, and qRT-PCR was used to detect the expression of CASC9. Two cell lines were transfected with Si-HIF-1α or Si-NC, followed by incubation under the 1% hypoxic condition for 9 h, and (C) the protein expression of p-AKT and HIF-1α was determined by western blot analysis; (D) the mRNA level of CASC9 was measured by qRT-PCR. (E) The putative binding sites of HIF-1α to the promoter of CASC9 are shown. (F) After treatment with 1% hypoxia for 9 h, a ChIP assay was carried out to determine the changes in binding abilities of different putative binding sites. The graphs shown are representative results from three independently repeated experiments. The mRNA value was normalized to GAPDH. **, P < 0.01; ***, P < 0.001.

Article Snippet: After treatment, approximately 4 × 10 6 SW1990 cells (suspended in 0.2 mL of PBS) were injected into the lateral tail vein of athymic mice (7 to 8 weeks old; HFK Bioscience Co.; n = 5 per group).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Binding Assay

Journal: Oncology Letters

Article Title: SREBP1 silencing inhibits the proliferation and motility of human esophageal squamous carcinoma cells via the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11853

Figure Lengend Snippet: Effects of SREBP1 on proliferation and epithelial-mesenchymal transition process. (A) After the silencing of SREBP1 by shRNA or transfection of the SREBP1 expression vector in ECA-109 or KYSE-150 cells for 24, 48 and 72 h, cell viability was examined using a CCK-8 assay. Data were analyzed using one-way ANOVA and Tukey's post hoc test. Expression levels of Ki-67 were analyzed in SREBP1 shRNA-expressing ECA-109 cells or SREBP1-overexpressing KYSE-150 cells at 48 h using (B) western blotting and (C) immunofluorescence staining (scale bars, 50 µm). (D) Representative blots showing the protein expression level changes of E-cadherin, N-cadherin and Vimentin from SREBP1-knockdown and related control ECA-109 cells, and SREBP1-overexpressing and related control KYSE-150 cells. β-actin served as the internal control. (E) Expression levels of Snail1 and Slug in ECA-109 and KYSE-150 cells treated with control, negative control of sh-SREBP1 for 48 h analyzed using reverse transcription-quantitative PCR. Data were analyzed using one-way ANOVA and Tukey's post hoc test. (F) Immunofluorescence staining showing the expression levels of E-cadherin (red), Vimentin (green) and DAPI in ECA-109 and KYSE-150 cells with different expression levels of SREBP1. Magnification, ×200. Scale bars, 50 µm. *P<0.05, **P<0.01, ***P<0.001. SREBP1, sterol regulatory element-binding protein 1; sh, small hairpin.

Article Snippet: ESCC cell lines TE-1, ECA-109 and KYSE-150 were purchased from Shanghai GeneChem Co., Ltd., which were authenticated using Short Tandem Repeat profiling.

Techniques: shRNA, Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Western Blot, Immunofluorescence, Staining, Negative Control, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Oncology Letters

Article Title: SREBP1 silencing inhibits the proliferation and motility of human esophageal squamous carcinoma cells via the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11853

Figure Lengend Snippet: Effects of SREBP1 on ESCC proliferation and metastasis are mediated by activating the Wnt/β-catenin signaling pathway. (A) mRNA and (B) protein expression levels of MMPs and VEGFs, including MMP2, MMP9, VEGF-A and VEGF-C were examined in SREBP1-downregulated or -upregulated ESCC cells using reverse transcription-quantitative PCR and ELISA assays. Data were analyzed using one-way ANOVA and Tukey's post hoc test. The effects of SREBP1-knockdown or overexpression on the (C) migration and (D) invasion, migration of ESCC cells were assessed by wound healing (magnification, ×100) and Transwell assays (magnification, ×200), respectively. Data were analyzed using unpaired Student's t-tests. (E) Effects of SREBP1-depletion and SREBP1-overexpression on the Wnt/β-catenin signaling pathway were determined using western blotting. The protein expression levels of Wnt/β-catenin pathway target proteins cyclin D1 and CD44 were also measured. β-actin and Lamin B1 served as the loading controls. (F) ECA-109-siSREBP1 cells were treated with or without 10 µM ICG-001, an inhibitor of the Wnt/β-catenin signaling pathway, and the expression levels of Wnt/β-catenin pathway target proteins and Ki-67 were examined using western blotting. (G) A Cell Counting Kit-8 assay was used to analyze cell viability of KYSE-150 and SREBP1-overexpressing KYSE-150 cells treated with escalating doses of ICG-001. Data were analyzed using unpaired Student's t-tests. *P<0.05, **P<0.01, ***P<0.001 vs. vector. SREBP1, sterol regulatory element-binding protein 1; ESCC, esophageal squamous cell carcinoma; sh, small hairpin; si, small interfering; p, phosphorylated; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; TCF1, T-cell factor 1; LEF1, lymphoid enhancer factor 1; NC, negative control.

Article Snippet: ESCC cell lines TE-1, ECA-109 and KYSE-150 were purchased from Shanghai GeneChem Co., Ltd., which were authenticated using Short Tandem Repeat profiling.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Over Expression, Migration, Western Blot, Cell Counting, Plasmid Preparation, Binding Assay, Negative Control

Journal: Oncology Letters

Article Title: SREBP1 silencing inhibits the proliferation and motility of human esophageal squamous carcinoma cells via the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11853

Figure Lengend Snippet: Silencing of SCD1 or blocking SREBP1/SCD1 desensitizes the Wnt/β-catenin signaling in ESCC cells. (A) Expression levels of SCD1 were assessed in cell lines treated with sh-SREBP1 or SREBP1-overexprssion vector. (B) Western blotting of the proteins involved in Wnt/β-catenin activation in KYSE-150 cells overexpressing SREBP1 or following transfection with siRNA-SCD1 or siRNA-NC for 24 h. (C) Western blotting showing the translocations of SREBP1 and SCD1, and Wnt/β-catenin pathway activation in ESCC cells treated with or without Fatostatin (10 µM) for 24 h. (D) Proliferation was assessed in cell lines treated with escalating doses of Fatostatin. Data were analyzed using unpaired Student's t-tests. *P<0.05, ***P<0.001 vs. vector. (E) Expression levels of VEGF-A and MMP9 were assessed in KYSE-150 cells treated with Fatostatin (10 µM) for 48 h. Data were analyzed using unpaired Student's t-tests. **P<0.01, ***P<0.001. SREBP1, sterol regulatory element-binding protein 1; SCD1, stearoyl-CoA desaturase 1; ESCC, esophageal squamous cell carcinoma; sh, small hairpin; si, small interfering; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; mSREBP1, mature sterol regulatory element-binding protein 1.

Article Snippet: ESCC cell lines TE-1, ECA-109 and KYSE-150 were purchased from Shanghai GeneChem Co., Ltd., which were authenticated using Short Tandem Repeat profiling.

Techniques: Blocking Assay, Expressing, Plasmid Preparation, Western Blot, Activation Assay, Transfection, Binding Assay