Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: siRNAs and their target genes used in the study

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Sequencing

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: TOP2A is involved in drug resistance to regorafenib. (A) Kaplan-Meier curves for HCC patients with low vs. high expression of TOP2A using TCGA database. (B, C) TOP2A expression in sorafenib-resistant cell lines and corresponding parental cell lines detected by Western blot and normalized to β-actinin (B); or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (C). (D) Huh7 and HepG2 cells were incubated in gradually increased concentrations of sorafenib for 48 h. Expression of TOP2A was detected by Western blot. Density of each band was normalized to that of β-actin. (E-H) Sorafenib-resistant Huh7-SR and HepG2-SR cells and corresponding parental cells were incubated with 0, 2.5, 5, 7.5 μM regorafenib for 48 h. The protein expression profiles were detected by Western blot. Density of each band was normalized to that of β-actin (E, F). Sorafenib-resistant HCC cells and parental cells were incubated with gradually increased concentrations of regorafenib or sorafenib for 48 h. TOP2A mRNA levels were measured by qRT-PCR and normalized against GAPDH. The relative TOP2A mRNA levels of cells treated with 0 μM sorafenib or regorafenib were normalized to 1 (G, H). *P < 0.05; **P < 0.01; and ***P < 0.001 vs. the sorafenib or regorafenib untreated cells.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Expressing, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Incubation

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: TOP2A silencing enhances sensitivity to regorafenib in sorafenib-resistant HCC cells. A, B. Sorafenib-resistant Huh7-SR and HepG2-SR cells were transfected with siTOP2A or negative control (NC) for 48 h. The corresponding un-transfected cells served as control. The protein expression profiles were detected by Western blot. Density of each band was normalized to that of β-actin. C, D. Huh7-SR and HepG2-SR cells and corresponding parental cells were transfected with siTOP2A for 24 h; subsequently incubated with increasing concentrations of regorafenib (Reg) for 48 h. Viability of transfected cells was normalized to control. E, F. Sorafenib-resistant cells and parental cells were exposed to 7.5 μM regorafenib for different periods, after transfected with siTOP2A. Cell viability was normalized with corresponding control. G, H. Huh7-SR and HepG2-SR cells were transfected with siTOP2A for 24 h; subsequently incubated with 7.5 μM regorafenib for 48 h. Protein expression profiles were detected by Western blot. Density of each band was normalized to that of β-actin. *P < 0.05; **P < 0.01 compared siTOP2A or regorafenib to control. #P < 0.05; ##P < 0.01 compared with regorafenib treated cells. †P < 0.05; ††P < 0.01 compared with siTOP2A treated cells.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Transfection, Negative Control, Control, Expressing, Western Blot, Incubation

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: Inhibition of TOP2A synergizes with regorafenib to suppress cell viability, promote apoptosis, and strengthen sensitivity of sorafenib-resistant HCC cells to regorafenib in vitro. (A-D) HepG2-SR and Huh7-SR as well as corresponding parental cells were exposed to different concentrations of doxorubicin (Dox) or/and regorafenib (Reg) for 48 h (A, B); or incubated for different periods in the presence or absence of regorafenib (7.5 μM) and doxorubicin (160 nM) (C, D). Cell viability (%) was compared with control. (E) Huh7-SR and HepG2-SR cells were incubated with 7.5 μM regorafenib or/and 160 nM doxorubicin for 48 h. The protein expression profile of cyclin D1 and cleaved caspase-3 was detected by Western blot. Density of each band was normalized to that of β-actin. (F) Migration of sorafenib-resistant HCC cells was examined by Wound healing assay (100 ×). (G) Transwell assays to examine synergistic effects of doxorubicin with regorafenib on migration and invasion (100 ×). (H) Synergistic effects of doxorubicin and regorafenib on 3D invasion (40 ×). *P < 0.05; **P < 0.01; indicating a significant difference from untreated cells. #P < 0.05; ##P < 0.01 compared with regorafenib treatment. †P < 0.05; ††P < 0.01 compared with doxorubicin treated cells.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Inhibition, In Vitro, Incubation, Control, Expressing, Western Blot, Migration, Wound Healing Assay

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: Inhibition of TOP2A synergizes with regorafenib to enhance the antitumor activity of regorafenib in vivo. (A-C) Subcutaneous xenografts were established. Mice received different treatments for 15 days. (A) Tumor images. (B) Tumor volume (mm3) was recorded. (C) Tumors were harvested and weighed. (D, E) Expression of cyclin D1 and cleaved caspase-3 by Western blot. Density of each band was normalized to that of β-actin (E). (F-I) Hematoxylin and eosin (H&E) (magnification, upper × 100, lower × 400) and immunohistochemistry staining (magnification, × 200). *P < 0.05; and **P < 0.01. #P < 0.05; ##P < 0.01 compared with regorafenib group. †P < 0.05; ††P < 0.01 compared with doxorubicin treated group.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Inhibition, Activity Assay, In Vivo, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: Schematic diagram depicting hypothesized roles of TOP2A in acquired resistance to regorafenib in HCC. →, positive regulation; ⊥, negative regulation or blockade.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques:

Journal: Oncotarget

Article Title: Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity

doi: 10.18632/oncotarget.23543

Figure Lengend Snippet: ( A ) Heat map for the changes of gene expression in human primary cardiomyocytes treated with trastuzumab, trastuzumab plus pertuzumab or T-DM1. Data with opposite influence on gene expression among these three treatments were removed, and then were further selected with threshold +/– 1.2 fold changes and P < 0.05 as selection criteria. This results in 2383 genes, which were input into the website Morpheus to generate a heat map according to the instruction. Following parameters were used to generate the heatmap with hierarchical clustering (metric: euclidean distance; linkage method: average). ( B ) Number of genes either up- or down- regulated by trastuzumab, trastuzumab plus pertuzumab or T-DM1. ( C ) Gene expression changes in DNA topoisomerase IIA (TOP2A) and IIB (TOP2B) in human primary cardiomyocytes treated by T-DM1, trastuzumab, or trastuzumab plus pertuzumab. ( D ) Gene clusters affected by trastuzumab treatment in human primary cardiomyocyte.

Article Snippet: Antibody against TOP2A was obtained from Origene.

Techniques: Expressing, Selection

Journal: Oncotarget

Article Title: Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity

doi: 10.18632/oncotarget.23543

Figure Lengend Snippet: ( A ) Human primary cardiomyocytes were treated with trastuzumab, pertuzumab, trastuzumab plus pertuzumab, ramucirumab and cetuximab at 50 µg/ml for 24 h. After treatments, Western blotting was performed, and expression levels of TOP2B and TOP2A were detected using antibodies directed against TOP2B or TOP2A. Equal loading of protein samples were confirmed by actin blots. Upper two panels: TOP2B expression; lower two panels: TOP2A expression. ( B ) TOP2B protein levels in human primary cardiomyocytes that were either treated with trastuzumab at 50 µg/ml (left graph) or doxorubicin (Dox) at 0.5 µM (right graph) or left untreated for 24 h. TOP2B expression was monitored using flow cytometry. ( C ) Quantitative analysis of fluorescent intensity obtained from flow cytometry analysis was presented in the form of bar graphs. Data are representative of two or more independent experiments and expressed as mean ± SEM. ( D ) TOP2B protein levels in human primary cardiomyocytes that were treated with trastuzumab (50 µg/ml), Dox (0.5 µM), trastuzumab (50 µg/ml) plus Dox (0.5 µM) or left untreated for 24h. TOP2B protein levels were monitored using flow cytometry. ( E ) TOP2B protein levels in human primary cardiomyocytes that were treated with Dox (0.5 µM) for 24 h or left untreated for 48 h. After 24h Dox treatment, Dox was removed from cell culture media, and cells were then either treated with trastuzumab (50 µg/ml) or left untreated for additional 24h. TOP2B protein levels were monitored using flow cytometry. The isotype in Figure and is a negative control. Experiments were performed in triplicates, and data are representative of two - three independent experiments. Student’s t -test was used for statistical significance. * P < 0.05; ** P < 0.01.

Article Snippet: Antibody against TOP2A was obtained from Origene.

Techniques: Western Blot, Expressing, Flow Cytometry, Cell Culture, Negative Control

Journal: Cell Division

Article Title: Anti-cancer effects of Coix seed extract through KCTD9-mediated ubiquitination of TOP2A in lung adenocarcinoma

doi: 10.1186/s13008-024-00112-2

Figure Lengend Snippet: KCTD9 affects the level of ubiquitination of TOP2A. a Interactions between TOP2A and TOP2B and KCTD9 in the String database. Protein expression of TOP2A ( b ) and TOP2B ( c ) in LUAD was analyzed in the UALCAN database. d Multiple ubiquitination modification sites are present in TOP2A in the GPS-Uber database. e The mRNA expression of KCTD9 in LUAD cells after infection of sh-NC or sh-KCTD9 was verified using RT-qPCR. f The mRNA expression of TOP2A in LUAD cells after infection of sh-NC or sh-KCTD9 was verified using RT-qPCR assays. g The protein expression of TOP2A in LUAD cells after infection of sh-NC or sh-KCTD9 was verified using western blot assays. The expression of TOP2A in A549 ( h ) and HCC827 ( i ) cells after infection of sh-NC or sh-KCTD9 in the presence of CHX. j Effect of KCTD9 on TOP2A ubiquitination detected by immunoprecipitation. k The protein expression of TOP2A in LUAD cells after infection of sh-NC or sh-KCTD9 with the proteasome inhibitor MG132 treatment. Data are presented as the mean ± SD of results from at least three independent experiments performed in triplicates, two-way ANOVA with Tukey’s posttest, * p < 0.05

Article Snippet: The supernatant was immunoprecipitated with protein magnetic beads, which were incubated with the antibody against TOP2A (1:200, ab12318, Abcam).

Techniques: Expressing, Modification, Infection, Quantitative RT-PCR, Western Blot, Immunoprecipitation

Journal: Cell Division

Article Title: Anti-cancer effects of Coix seed extract through KCTD9-mediated ubiquitination of TOP2A in lung adenocarcinoma

doi: 10.1186/s13008-024-00112-2

Figure Lengend Snippet: Silencing of TOP2A reverses the effects of KCTD9 knockdown on LUAD cell immune escape. a Correlation of TOP2A expression in LUAD with human CD8 + T cell infiltration predicted in the TIMER database. b Correlation between TOP2A and PD-L1 expression predicted in the GEPIA database. c The prognostic outcomes in patients with high or low TOP2A expression are predicted in the Kaplan–Meier Plotter database. d The mRNA expression of TOP2A in LUAD cells after infection of sh-NC or sh-TOP2A was determined using RT-qPCR. e The mRNA expression of PD-L1 in LUAD cells was detected using RT-qPCR. f The protein expression of PD-L1 in LUAD cells was detected using western blot assays. g - j The levels of TNF-α, IFN-γ, CXCL10, and CXCL9 in the co-culture system of LUAD cells and human CD8 + T cells. k Proliferation of human CD8 + T cells in co-culture system detected by CFSE assays. l The ability of differently treated LUAD cells against human CD8 + T cells was evaluated using tumor cell killing assay. Data are presented as the mean ± SD of results from at least three independent experiments performed in triplicates, two-way ANOVA with Tukey’s posttest, * p < 0.05

Article Snippet: The supernatant was immunoprecipitated with protein magnetic beads, which were incubated with the antibody against TOP2A (1:200, ab12318, Abcam).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Co-Culture Assay

Journal: Cell Division

Article Title: Anti-cancer effects of Coix seed extract through KCTD9-mediated ubiquitination of TOP2A in lung adenocarcinoma

doi: 10.1186/s13008-024-00112-2

Figure Lengend Snippet: The inhibiting effects of CSE on the immune escape of LUAD cells in vivo are reversed by KCTD9 knockdown. a Tumor volume in mice in 3 weeks. b Weight changes in mouse tumors. c The protein expression of PD-L1 in mouse tumor tissues was examined using western blot assays. d Immunofluorescence analysis of CD8 + T cell infiltration in mouse tumor tissues. e The levels of CD8 + T cell activation markers GzmB and Perforin in the supernatant of the co-culture system were detected by ELISA. f Detection of apoptosis in mouse tumor tissues by TUNEL assay. The levels of TNF-α ( g ), IFN-γ ( h ), CXCL10 ( i ), and CXCL9 ( j ) in the mouse tumor tissues by ELISA. ( k ) The mRNA expression of KCTD9 in mouse tumor tissues was assessed by RT-qPCR. l The protein expression of TOP2A in mouse tumor tissues was assessed by western blot assays. Data are presented as the mean ± SD of results from at least three independent experiments performed in triplicates, one-way or two-way ANOVA with Tukey’s posttest, * p < 0.05

Article Snippet: The supernatant was immunoprecipitated with protein magnetic beads, which were incubated with the antibody against TOP2A (1:200, ab12318, Abcam).

Techniques: In Vivo, Expressing, Western Blot, Immunofluorescence, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Quantitative RT-PCR