Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: p53 signaling pathway was the major pathway regulated by Z-7 in p53 WT CRC cells. ( a ) Volcano plot of DEGs from RNA-seq of HCT116 cells treated with Z-7; ( b ) KEGG pathway enrichment analysis showed the DEGs were enriched in the p53 pathway and other cell cycle related pathways; ( c ) Heatmap of p53 pathway related genes expression in HCT116 treated with Z-7; ( d ) Heatmap of selected differentially expressed genes in both HCT116 and HT29 cells treated with Z-7; ( e-g ) qPCR analysis of p53 down-stream genes CDKN1A ( e ), MDM2 ( f ), and BBC3 ( g ) in Z-7 treated HCT116 cells; ( h ) KEGG pathway enrichment analysis showed the differential expressed proteins were enriched in the p53 signaling pathway and cell cycle related pathways; ( i ) Heatmap of p53 pathway related proteins expression in HCT116 treated with Z-7; ( j ) Cell cycle analysis of LoVo treated with different concentrations of Z-7 for 24 hours; ( k ) Quantification of Annexin V + cells percentage in LoVo treated with different concentrations of Z-7; ( l ) Western blot analysis of apoptotic markers in LoVo cells treated different concentrations of with Z-7 for 24 hours. CP, cellular process; EIP, encrionmental information processing; Meta, metabolism; OS, organismal systems. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The IHC protocol has been thoroughly described in our previous study [ ]. p53 (7F5) Rabbit antibody (2527S, CST) was used.

Techniques: RNA Sequencing, Expressing, Cell Cycle Assay, Western Blot

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: Z-7 inhibited proliferation of p53 WT CRC cells through activation of the p53 signaling pathway. ( a-b ) Western blot analysis of p53 and p21 expression in HCT116 ( a ) and LoVo ( b ) after varied concentrations of Z-7 treatment; ( c-f ) qPCR analysis of TP53 ( c ) and down-stream genes CDKN1A ( d ), MDM2 ( e ), and BBC3 ( f ) in TP53 knockdown HCT116 cells after varied concentrations of Z-7 treatment; ( g ) Western blot analysis of apoptotic markers, p53, and p21 in TP53 knockdown HCT116 cells after varied concentrations of Z-7 treatment; ( h ) Quantification of Annexin V + cells percentage in TP53 knockdown HCT116 cells after varied concentrations of Z-7 treatment; ( i ) Cell cycle analysis of TP53 knockdown HCT116 treated with 2.5 µM Z-7. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The IHC protocol has been thoroughly described in our previous study [ ]. p53 (7F5) Rabbit antibody (2527S, CST) was used.

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Cell Cycle Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: Z-7 restored p53 protein level through MDM2 mediated p53 ubiquitination by inhibiting E3 ligase function of MDM2. ( a ) Cyclohexamide (CHX) chasing assay showed Z-7 stabilizing p53 protein level; ( b ) Statistical data of two independent CHX chasing experiments was generated by using grayscale analysis in ImageJ; ( c ) Western blot analysis showed over expressing MDM2 can reverse the effects of Z-7 on p53 accumulation; ( d ) Western blot analysis showed Z-7 down regulated the MDM2 mediated ubiquitin level of p53; ( e ) The structure of Z-7 tagged with biotin label at the end of acetyl group (named Biotin-Z-7); ( f ) Competition assay showed the Z-7 physically interacted with MDM2. Whole cell lysis (WCL) of HEK-293T cells with over-expressed FLAG-MDM2 was incubated with or without 100 µM of Z-7 first, and then incubated with streptavidin beads coated with different concentrations of Biotin-Z-7, M for marker; ( g ) Co-IP showed Z-7 did not interrupt the MDM2-p53 interaction. Briefly, FLAG-MDM2 and Myc-p53 were co-transfected into 293T cells, followed by treatment with or without Z-7. Nutlin-3A served as a positive control. Samples were harvested after treatment, and subjected to immunoprecipitation using an anti-FLAG antibody. ( h ) Schematic overview of the truncated MDM2 fragments; ( i ) Pulldown assay showed the Z-7 bound to the RING domain of MDM2. WCL of HEK-293T cells with over-expressed truncated FLAG-MDM2 fragments were incubated with streptavidin beads coated with DMSO or 25 µM of Z-7; ( j ) Western blot analysis showed Z-7 inhibited MDM2 auto-ubiquitination. FLAG-MDM2 and HA-Ub were co-transfected into 293T cells following Z-7 treatment for 24 hours. Before sampls were harvested, samples were treated with or with 10 µM of MG-132 for 6 hours. Then samples were subjected to ubiquitin analysis and western blot analysis. Statistical significance is indicated as *, p < 0.05

Article Snippet: The IHC protocol has been thoroughly described in our previous study [ ]. p53 (7F5) Rabbit antibody (2527S, CST) was used.

Techniques: Ubiquitin Proteomics, Generated, Western Blot, Expressing, Competitive Binding Assay, Lysis, Incubation, Marker, Co-Immunoprecipitation Assay, Transfection, Positive Control, Immunoprecipitation

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: Z-7 suppressed the growth of HCT116 xenograft model in mice. ( a ) Schematic overview and timeframe of the animal experiments. BALB/c nude mice were first inoculated subcutaneously with HCT116 cells. From day 6, control or Z-7 was administered via intratumorally injection. All mice were sacrificed on day 16 and the xenografts were excised for further analysis; ( b ) HCT116 xenograft tumor growth curves of intra-tumoral Z-7 administration in nude mice compared with control group; ( c ) The weight of HCT116 xenograft tumors in Z-7 treatment group compared with control group; ( d ) Representative image of HCT116 xenograft tumors with or without Z-7 treatment in nude mice; ( e ) Western blot analysis of p53 and p21 in HCT116 xenograft tumor treated with or without Z-7 in nude mice. 50 mg of sample was excised from each xenograft tumor, then homogenized and lysed with Laemmli lysis buffer, and subjected to immunoblotting analysis; ( f ) Immunohistochemistry analysis of p53 expression level in HCT116 xenograft tumor treated with or without Z-7; ( g ) Proposed model. Z-7 may inhibit MDM2 E3 ligase function, resulting downregulation of p53 ubiquitination level, thereby stabilizing p53 protein and activating relevant downstream proteins in the p53 signaling pathway, eventually leading to inhibit the colorectal cancer progression. Moreover, Z-7 also can increase the ROS level which also promotes cell cycle arrest and apoptosis in colorectal cancer. All results are shown as mean±S.E.M ( n = 5). * p < 0.05, ** p < 0.01，*** p < 0.001

Article Snippet: The IHC protocol has been thoroughly described in our previous study [ ]. p53 (7F5) Rabbit antibody (2527S, CST) was used.

Techniques: Control, Injection, Western Blot, Lysis, Immunohistochemistry, Expressing, Ubiquitin Proteomics