Journal: Experimental hematology & oncology

Article Title: p53 directly downregulates the expression of CDC20 to exert anti-tumor activity in mantle cell lymphoma.

doi: 10.1186/s40164-023-00381-7

Figure Lengend Snippet: Fig. 2 CDC20 mRNA expression was associated with the therapeutic effect and prognosis of MCL patients. Among 24 MCL patients with PBMCs extracted, CDC20 mRNA expression was significantly different in the subgroups of treatment response (A), MIPI score (B), and MIPI-c score (C). A Patients were divided into the CR/PR group and the PD/SD group according to treatment response, and CDC20 mRNA expression level between the two groups was compared. B Patients were divided into the low risk group, the intermediate risk group and the high risk group according to MIPI score, and CDC20 mRNA expression level among the three groups was compared. C Patients were divided into the low risk group, low-intermediate risk group, high-intermediate risk group, and the high risk group according to MIPI-c score, and CDC20 mRNA expression level among the four groups was compared. D The relationship between CDC20 expression level and overall survival of MCL patients was analyzed via GSE93291 dataset (n = 123). *P < 0.05, **P < 0.01, ***P < 0.001, ns meant P > 0.05

Article Snippet: Cells were exposed to designated concentrations of CDC20 inhibitor apcin (Selleck, USA) or p53 agonist nutlin-3a (Selleck, USA) alone, or co-treated with apcin and nutlin-3a, and incubated for 24 h, 48 h and 72 h. Nutlin-3a could inhibit the interaction between MDM2 and p53 and activate p5333, so we identified nutlin-3a as the p53 agonist in this study.

Techniques: Expressing

Journal: Experimental hematology & oncology

Article Title: p53 directly downregulates the expression of CDC20 to exert anti-tumor activity in mantle cell lymphoma.

doi: 10.1186/s40164-023-00381-7

Figure Lengend Snippet: Fig. 6 p53 transcriptionally repressed CDC20 expression by directly binding to CDC20 promoter. A Dual-luciferase reporter gene assay was conducted to verify whether p53 could regulate CDC20 promoter activity (TSS-1495 bp ~ + 26 bp). The results implied that p53 overexpression in 293 T cells could significantly inhibit CDC20 promoter activity. Relative luciferase activity was expressed as the ratio of firefly luciferase activity to renilla luciferase activity. Data were obtained from three independent experiments and presented as mean ± SD. ***P < 0.001. B CUT&Tag assay proved that p53 directly bound to CDC20 promoter region at TSS-492 bp ~ + 101 bp. Compared with the untreated control group, CDC20 promoter signal was significantly decreased in Z138 cells treated with 5 μM nutlin-3a for 24 h. The red box indicated the binding site of p53 on CDC20 promoter on chromosome 1, and the characteristic signal peaks of the control group (upper) and nutlin3a-treated group (lower) in this region. C Z138 cells were pre-treated with 15 μM PFT-α for 1 h, and then co-treated with 50 μM apcin for 24 h. WB results showed pre-treatment of Z138 cells with PFT-α could rescue CDC20 expression reduced by apcin treatment. Data were obtained from at least three independent experiments and presented as mean ± SD. **P < 0.01, ***P < 0.001, ns meant P > 0.05

Article Snippet: Cells were exposed to designated concentrations of CDC20 inhibitor apcin (Selleck, USA) or p53 agonist nutlin-3a (Selleck, USA) alone, or co-treated with apcin and nutlin-3a, and incubated for 24 h, 48 h and 72 h. Nutlin-3a could inhibit the interaction between MDM2 and p53 and activate p5333, so we identified nutlin-3a as the p53 agonist in this study.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Gene Assay, Activity Assay, Over Expression, Control

Journal: Experimental hematology & oncology

Article Title: p53 directly downregulates the expression of CDC20 to exert anti-tumor activity in mantle cell lymphoma.

doi: 10.1186/s40164-023-00381-7

Figure Lengend Snippet: Fig. 11 Targeted protein expression of the four mice groups. A Representative IHC images of p53, CDC20, cleaved PARP and K-i67 staining of tumor tissues in the control group, the nutlin-3a group, the apcin group, and the nutlin-3a plus apcin group (× 200 magnification; scale bar, 100 μm). B The protein expression of p53, CDC20, cleaved PARP and Ki-67 of tumor tissues were determined by IHC quantitative analysis in the control group, the nutlin-3a group, the apcin group, and the nutlin-3a plus apcin group, which were calculated by the MOD value. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Cells were exposed to designated concentrations of CDC20 inhibitor apcin (Selleck, USA) or p53 agonist nutlin-3a (Selleck, USA) alone, or co-treated with apcin and nutlin-3a, and incubated for 24 h, 48 h and 72 h. Nutlin-3a could inhibit the interaction between MDM2 and p53 and activate p5333, so we identified nutlin-3a as the p53 agonist in this study.

Techniques: Expressing, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells

doi: 10.3390/ijms21186692

Figure Lengend Snippet: Cell division cycle 20 (CDC20) was overexpressed in human cancer cells and upregulated after radiation. ( A , B ) Transcriptional ( A ) and translational levels ( B ) of CDC20 in HIEC cells, HCT116 cells, and LOVO cells were detected by quantitative real-time PCR and immunoblotting. ( C , D ) HCT116 cells ( C ) and LOVO cells ( D ) were irradiated with different doses of γ-radiation, and the protein levels of CDC20 were analyzed by Western blotting 24 h later. ( E , F ) The expression of CDC20 in HCT116 cells ( E ) and LOVO cells ( F ) at different time points after 5 Gy γ-radiation. Actin was used as an internal control. Data were pooled from three independent experiments, and the results are represented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CDC20 inhibitor apcin was purchased from Tocris (Minneapolis, MN, USA).

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

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells

doi: 10.3390/ijms21186692

Figure Lengend Snippet: CDC20 attenuates the sensitivity of HCT116 cells to gamma-rays. ( A ) CDC20 was stably reduced in HCT116 cells by lentiviral infection. The expression levels of CDC20 were determined by immunoblotting analysis (sh-1/2/3 stands for three short hairpin RNA segments). ( B ) The expression levels of γH2AX and Rad51 were detected by Western blotting in control and CDC20 knockdown cells at 24 h after 5 Gy gamma-ray irradiation. ( C ) The expression level of Rad51 was detected in control and CDC20 overexpression cells at 24 h after 5 Gy gamma-ray irradiation. ( D ) A clongenic assay of HCT116 cells with reduced CDC20 expression and control vector cells was carried out after irradiation with different doses of gamma-rays. ( E ) HCT116 cells with reduced CDC20 expression and control vector cells were exposed with the indicated doses of gamma-ray irradiation, and cell viability was measured 24 h later. ( F , G ) Higher expression level of apaf1, cleaved caspase-9, cleaved caspase-7, and cleaved caspase-3 ( F ) and more robust caspase-3/7 activation ( G ) were detected in CDC20 knockdown HCT116 cells compared with control vector cells 24 h after 5 Gy gamma-ray irradiation. ( H ) HCT116 cells were pretreated with different doses of apcin (0–10 µM) for 24 h before 5 Gy gamma-ray irradiation, and cell viability was detected 24 h after irradiation. ( I ) Cells were preincubated with apcin (25 or 50 µM) for 24 h before 5 Gy gamma-ray irradiation; then, the expression levels of cleaved caspase-7 and cleaved caspase-3 were determined by Western blot. Actin was used as an internal control. Data were pooled from three independent experiments and the results are represented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CDC20 inhibitor apcin was purchased from Tocris (Minneapolis, MN, USA).

Techniques: Stable Transfection, Infection, Expressing, Western Blot, shRNA, Control, Knockdown, Irradiation, Over Expression, Plasmid Preparation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells

doi: 10.3390/ijms21186692

Figure Lengend Snippet: CDC20 inhibits radiation-induced apoptosis signals by interacting with Mcl-1. ( A ) The expression levels of Mcl-1, Bak, Bax, Puma, Bcl-2, and Bcl-xL were detected by immunoblotting analysis in CDC20 knockdown HCT116 cells compared with control vector cells 24 h after 5 Gy gamma-ray irradiation. ( B ) The expression level of Mcl-1 was determined by immunoblotting analysis in CDC20-overexpressing HCT116 cells compared with control vector cells 24 h after 5 Gy gamma-ray irradiation. ( C ) After irradiating with 5 Gy gamma-rays, the expression levels of CDC20 and Mcl-1 were detected by immunoblotting analysis at different time points. ( D ) Interaction of endogenous CDC20 with Mcl-1 was examined by Co-immunoprecipitation assay in HCT116 cells. ( E ) Purified Flag-Mcl-1 immobilized on beads was incubated with purified glutathione S-transferase (GST)-CDC20. Input and bead-bound proteins were analyzed by immunoblotting with anti-CDC20 antibody. Actin was used as an internal control. Data were pooled from three independent experiments and the results are represented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001. ns = non significant.

Article Snippet: The CDC20 inhibitor apcin was purchased from Tocris (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Knockdown, Control, Plasmid Preparation, Irradiation, Co-Immunoprecipitation Assay, Purification, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells

doi: 10.3390/ijms21186692

Figure Lengend Snippet: The radioresistant function of CDC20 was mediated by the Mcl-1/p-Chk1 signal axis. ( A – C ) The expression level of p-Chk1 was detected in Mcl-1 knockdown HCT116 cells ( A ), CDC20 knockdown HCT116 cells ( B ), and Mcl-1-overexpressing HCT116 cells ( C ). ( D ) HCT116 cells were pretreated with different doses of AZD7762 (0–100 nM) for 24 h before 5 Gy gamma-ray irradiation, and the expression levels of γH2AX, Rad51, and cleaved caspase-3 were detected 24 h after irradiation. ( E , F ) HCT116 cells were pretreated with different doses of AZD7762 (0–100 nM) for 24 h before 5 Gy irradiation; cell viability ( E ) and caspase-3/7 activity ( F ) were detected 24 h after 5 Gy γ-radiation. Actin was used as an internal control. Data were pooled from three independent experiments and the results are represented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CDC20 inhibitor apcin was purchased from Tocris (Minneapolis, MN, USA).

Techniques: Expressing, Knockdown, Irradiation, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells

doi: 10.3390/ijms21186692

Figure Lengend Snippet: CDC20 knockdown suppresses the tumor formation of colorectal cancer (CRC) through inducing apoptosis in vivo. ( A ) Plan of animal experiments. ( B ) Tumor xenografts of each group are shown. Each group of mice was composed of five BALB/c-nu/nu mice. HCT116 control vector cells (sh-C, 5 × 10 6 ) and CDC20 knockdown HCT116 cells (sh-CDC20, 5 × 10 6 ) were inoculated under the dorsal skin of BALB/c-nu/nu mice. After 15 days of injection, mice were irradiated with 0 Gy, 10 Gy or 15 Gy gamma-rays. On the 33rd day after injection, the mice were sacrificed, and tumors were obtained. ( C , D ) The tumor volume ( C ) and net weights ( D ) of each group are shown. Tumor size was measured with dull-edged vernier calipers every 3 days, calculated as follows: volume = (length × width 2 )/2. The weight of the tumor was determined when the mice were sacrificed. ( E ) Apoptotic cells in tumor sections were identified by Terminal dexynucleotidyl Transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. Representative images of tumor sections from sh-C and sh-CDC20 mice under different doses of gamma-ray treatment are shown. The scale bar represents 25 µm. ( F ) The number of apoptotic cells shown in ( E ) was quantified using GraphPad Prism. ( G ) Necrotic cells in tumor sections were identified by Hematoxylin-Eosin (H&E) staining. Representative images of tumor sections from sh-C and sh-CDC20 mice under different doses of gamma-ray treatment are shown. The scale bar represents 50 µm. ( H ) Immunohistochemical analysis of Mcl-1 and p-Chk1 expression in the tumor xenografts. Representative immunohistochemical staining of both Mcl-1 and p-Chk1 from sh-C and sh-CDC20 mice under different doses of gamma-ray treatment are shown (magnification, ×200). The scale bar represents 50 µm. ( I , J ) The number of positive cells shown in ( H ) was quantified using GraphPad Prism. Data were pooled from three independent experiments and the results are represented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The CDC20 inhibitor apcin was purchased from Tocris (Minneapolis, MN, USA).

Techniques: Knockdown, In Vivo, Control, Plasmid Preparation, Injection, Irradiation, End Labeling, TUNEL Assay, Staining, Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells

doi: 10.3390/ijms21186692

Figure Lengend Snippet: Schematic representation of the mechanism of CDC20-mediated radiosensitization of CRC cells.

Article Snippet: The CDC20 inhibitor apcin was purchased from Tocris (Minneapolis, MN, USA).

Techniques: