Journal: Clinical and Translational Medicine

Article Title: β‐elemene promotes ferroptosis to improve the sensitivity of imatinib in gastrointestinal stromal tumours by targeting N6AMT1

doi: 10.1002/ctm2.70438

Figure Lengend Snippet: β‐elemene induces ferroptosis in imatinib‐resistant GIST cells. (A) Chemical structure of β‐elemene. (B‐C) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and GSEA showed that genes involved in ferroptosis were significantly dysregulated under β‐elemene treatment. (D) The Fe 2+ levels detected by FerroOrange fluorescence probe in GIST‐882‐IR and GIST‐T1‐IR cells treated with DMSO, imatinib, β‐elemene or imatinib+β‐elemene for 24 h. (E) Quantification of relative fluorescence intensity of Fe 2+ levels by Image J. (F) The MDA levels detected in GIST‐882‐IR and GIST‐T1‐IR cells treated with DMSO, imatinib, β‐elemene or imatinib+β‐elemene for 24 h. (G) Representative fluorescent images of lipid peroxidation detected by BODIPY (581/591) C11 probe in GIST‐882‐IR and GIST‐T1‐IR cells treated with DMSO, imatinib, β‐elemene or imatinib+β‐elemene for 24 h. Scale bar = 100 µm. (H) Quantification of relative fluorescence intensity of oxidised BODIPY by Image J. (I) The ROS accumulation was detected using flow cytometry analysis and statistical histograms of positive cells in GIST‐882‐IR and GIST‐T1‐IR cells treated with DMSO, imatinib, β‐elemene or imatinib+β‐elemene for 24 h. (J) Representative cell ultrastructural images of GIST‐T1‐IR cells treated with DMSO, imatinib, β‐elemene or imatinib+β‐elemene for 24 h. All experiments were performed in triplicate. Data represent the mean ± SD; * p < .05; ** p < .01; *** p < .001. An unpaired t ‐test was used unless otherwise stated.

Article Snippet: All cell lines were cultured in an incubator with 37 ° C and 5% CO 2 . β‐elemene was provided by Dalian HolleyKingkong Pharmaceutical Co. Ltd. Imatinib (T6230) was purchased from TargetMol.

Techniques: Fluorescence, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: β‐elemene promotes ferroptosis to improve the sensitivity of imatinib in gastrointestinal stromal tumours by targeting N6AMT1

doi: 10.1002/ctm2.70438

Figure Lengend Snippet: β‐elemene promotes imatinib sensitivity in imatinib‐resistant GISTs through ferroptosis. (A) The interaction between imatinib and β‐elemene on cell cytotoxicity was examined by the median‐effect method of Chou–Talalay. (B) The dose and combination index of imatinib in combination with β‐elemene on GIST‐882‐IR and GIST‐T1‐IR cells was estimated by calculation of combination index (CI) values using Compusyn software. The darker point indicates a stronger synergistic effect. The fraction affected values indicate the percentage of cell inhibition, while the CI values indicate the effects of combination treatments. (C) Antitumour effects of imatinib and β‐elemene in GIST‐882‐IR and GIST‐T1‐IR cells after the indicated treatment were evaluated by colony formation assay. (D) Statistical analysis of the colony formation assay. (E) Representative results of PI‐positive cells in IM‐exposed GIST‐882‐IR and GIST‐T1‐IR after the indicated treatment and quantitative analysis after the treatment for 48 h. (F) Statistical histogram of the flow cytometry cell death analysis. (G) Antitumour effects of GIST‐882‐IR and GIST‐T1‐IR cells treated with or without ferroptosis inhibitor Ferrostatin‐1 (Fer‐1) were evaluated by colony formation assay. (H) Statistical analysis of the colony formation assay of GIST‐882‐IR and GIST‐T1‐IR cells treated with or without ferroptosis inhibitor Fer‐1. (I) Cell viability of GIST‐882‐IR and GIST‐T1‐IR cells treated with or without ferroptosis inhibitor Fer‐1. (J) Schematic description of the in vivo anticancer effect of combined treatment with β‐elemene and imatinib in the cell line‐based xenograft model. (K) Photograph and comparison of tumour sizes in the indicated groups. (L) Growth curve of GIST‐T1‐IR xenografts in the indicated groups. (M) The tumour weight of GIST‐T1‐IR xenografts in the indicated groups. (N) Representative images of IHC staining of MKI67/K‐67 in mouse tumours. All experiments were performed in triplicate. Data represent the mean ± SD; * p < .05; ** p < .01; *** p < .001. An unpaired t ‐test was used unless otherwise stated.

Article Snippet: All cell lines were cultured in an incubator with 37 ° C and 5% CO 2 . β‐elemene was provided by Dalian HolleyKingkong Pharmaceutical Co. Ltd. Imatinib (T6230) was purchased from TargetMol.

Techniques: Software, Inhibition, Colony Assay, Flow Cytometry, In Vivo, Comparison, Immunohistochemistry

Journal: Clinical and Translational Medicine

Article Title: β‐elemene promotes ferroptosis to improve the sensitivity of imatinib in gastrointestinal stromal tumours by targeting N6AMT1

doi: 10.1002/ctm2.70438

Figure Lengend Snippet: β‐elemene induced ferroptosis in imatinib‐resistant GISTs through HMOX1. (A) Heatmap of the RNA‐seq analysis results for GIST‐T1‐IR cells treated with DMSO or β‐elemene. (B) Volcano plot of down‐regulated and upregulated for GIST‐T1‐IR cells treated with DMSO or β‐elemene. (C) Western blotting analysis of HMOX1 and GPX4 expression in GIST cells treated with imatinib, β‐elemene or imatinib+β‐elemene. (D) Western blotting analysis of HMOX1 expression in parental and imatinib‐resistant GIST cells. (E) Western blotting analysis of HMOX1 in GIST‐882‐IR and GIST‐T1‐IR cells transfected with HMOX1‐expressing plasmid. (F) CCK‐8 assay of sensitivity to imatinib in HMOX1‐overexpressed GIST‐882‐IR and GIST‐T1‐IR cells versus control GIST‐882_IR and GIST‐T1‐IR cells. (G) The colony formation assay of sensitivity to imatinib in HMOX1‐overexpressed GIST‐882‐IR and GIST‐T1‐IR cells versus control GIST‐882_IR and GIST‐T1‐IR cells. (H) Western blot showing changes in HMOX1 expression in response to Hemin. (I‐J) The colony formation assay and statistical histogram of GIST‐882‐IR and GIST‐T1‐IR cells treated with imatinib+β‐elemene with or without hemin. (K) Cell death measurements by flow cytometry analysis in GIST‐882‐IR and GIST‐T1‐IR cells treated with imatinib+β‐elemene with or without hemin. (L–M) The colony formation assay and statistical histogram of GIST‐882‐IR and GIST‐T1‐IR cells treated with imatinib+β‐elemene with or without zinc protoporphyrin‐9 (ZnPP). (N) Cell death measurements by flow cytometry analysis in GIST‐882‐IR and GIST‐T1‐IR cells treated with imatinib+β‐elemene with or without ZnPP. (O) Photograph and comparison of tumour sizes in different groups. (P) Growth curve of GIST‐T1‐IR xenografts in the indicated groups. (Q) The tumour weight of GIST‐T1‐IR xenografts in the indicated groups. All experiments were performed in triplicate. Data represent the mean ± SD; * p < .05; ** p < .01; *** p < .001. An unpaired t ‐test was used unless otherwise stated.

Article Snippet: All cell lines were cultured in an incubator with 37 ° C and 5% CO 2 . β‐elemene was provided by Dalian HolleyKingkong Pharmaceutical Co. Ltd. Imatinib (T6230) was purchased from TargetMol.

Techniques: RNA Sequencing, Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Control, Colony Assay, Flow Cytometry, Comparison

Journal: Clinical and Translational Medicine

Article Title: β‐elemene promotes ferroptosis to improve the sensitivity of imatinib in gastrointestinal stromal tumours by targeting N6AMT1

doi: 10.1002/ctm2.70438

Figure Lengend Snippet: β‐elemene targets N6AMT1 to promote imatinib sensitivity in imatinib‐resistant GIST cells via the nuclear factor erythroid 2‐related factor 2 (NRF2)/HMOX1 axis. (A) The workflow for cellular targets identification of β‐elemene in GIST‐T1‐IR by thermal proteome profiling (TPP). (B) Heatmap of differentially expressed protein in GIST‐T1‐IR cells between control and β‐elemene‐treated groups. (C) Venn diagram of β‐elemene target screening. (D) Volcano plot of cellular targets of β‐elemene by the TPP strategy. (E) Western blot analysis of NRF2 and HMOX1 in GIST cells with or without N6AMT1 knockdown by small interfering RNA (siRNA). (F) Western blot analysis of NRF2 and HMOX1 in GIST cells with or without N6AMT1 overexpression. (G) Cellular thermal shift assay demonstrated a stabilisation effect of N6AMT1 with β‐elemene. (H) The interaction between β‐elemene with N6AMT1 targeting Asp103 was predicted by molecular docking. (I) GIST‐T1‐IR cell lysates were incubated with either biotin or β‐elemene‐biotin at 4°C overnight, and then a pulldown assay was performed. (J) GIST‐T1‐IR cell lysates were preincubated with either DMSO or free β‐elemene, followed by subsequent incubation with β‐elemene‐biotin. The interaction between N6AMT1 and β‐elemene was then detected by capturing β‐elemene‐biotin. (K) The mutant N6AMT1 proteins were incubated with β‐elemene, followed by protein affinity pull‐down assay and detected by immunoblotting. (L) Western blot analysis of N6AMT1/NRF2/HMOX1 axis in GIST cells treated with imatinib, β‐elemene or imatinib+β‐elemene. (M) Western blot analysis of NRF2 in cytosol protein and nuclear protein. (N) The staining intensity and localisation of NRF2 in the indicated groups were analysed by immunofluorescence staining. (O) Western blot analysis of the relationship of NRF2 and HMOX1 in ferroptosis with knockdown of NRF2 in GIST cells followed by imatinib+β‐elemene treatment. (P) Methylation level of NRF2 promoter in GIST‐T1‐IR cell treated with imatinib, β‐elemene or imatinib+β‐elemene. (Q) Methylation level of NRF2 promoter in GIST‐T1‐IR cell with or without N6AMT1 knockdown by siRNA. (R) Methylation level of NRF2 promoter in GIST‐T1‐IR cell with or without N6AMT1 overexpression. Data represent the mean ± SD; * p < .05; ** p < .01; *** p < .001. An unpaired t ‐test was used unless otherwise stated.

Article Snippet: All cell lines were cultured in an incubator with 37 ° C and 5% CO 2 . β‐elemene was provided by Dalian HolleyKingkong Pharmaceutical Co. Ltd. Imatinib (T6230) was purchased from TargetMol.

Techniques: Control, Western Blot, Knockdown, Small Interfering RNA, Over Expression, Thermal Shift Assay, Incubation, Mutagenesis, Pull Down Assay, Staining, Immunofluorescence, Methylation

Journal: Clinical and Translational Medicine

Article Title: β‐elemene promotes ferroptosis to improve the sensitivity of imatinib in gastrointestinal stromal tumours by targeting N6AMT1

doi: 10.1002/ctm2.70438

Figure Lengend Snippet: β‐elemene improves imatinib therapeutic efficiency in imatinib‐resistant GIST. (A–D) IHC staining of Nrf2, HMOX1 and 4‐HNE in tumour tissues generated by GIST‐T1‐IR cells‐based xenograft in the indicated groups. Scale bars, 50 µm. (E) Schematic description of the in vivo anticancer effect of combined treatment with imatinib and β‐elemene in the patient‐derived xenograft (PDX) model. (F) Photograph and comparison of tumour sizes of the PDX model in the indicated groups. (G) Growth curve of the PDX model in the indicated groups. (H) The tumour weight of the PDX model in the indicated groups. (I–M) IHC staining of 4‐HNE and HMOX1 in tumour tissues generated by the PDX model in the indicated groups. Scale bars, 50 µm. Data represent the mean ± SD; * p < .05; ** p < .01; *** p < .001. An unpaired t ‐test was used unless otherwise stated.

Article Snippet: All cell lines were cultured in an incubator with 37 ° C and 5% CO 2 . β‐elemene was provided by Dalian HolleyKingkong Pharmaceutical Co. Ltd. Imatinib (T6230) was purchased from TargetMol.

Techniques: Immunohistochemistry, Generated, In Vivo, Derivative Assay, Comparison

Journal: Clinical and Translational Medicine

Article Title: β‐elemene promotes ferroptosis to improve the sensitivity of imatinib in gastrointestinal stromal tumours by targeting N6AMT1

doi: 10.1002/ctm2.70438

Figure Lengend Snippet: Summary diagram of the mechanism that β‐elemene increased the sensitivity of GIST cells to imatinib. β‐elemene specifically targets N6AMT1, inhibiting its transcriptional repression function and activating the NRF2‐HMOX1 signalling pathway to induce ferroptosis.

Article Snippet: All cell lines were cultured in an incubator with 37 ° C and 5% CO 2 . β‐elemene was provided by Dalian HolleyKingkong Pharmaceutical Co. Ltd. Imatinib (T6230) was purchased from TargetMol.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Anti-Lung-Cancer Activity and Liposome-Based Delivery Systems of β -Elemene

doi: 10.1155/2012/259523

Figure Lengend Snippet: Chemical structure of β -Elemene.

Article Snippet: Afterwards, an injection preparation containing 85% β -elemene developed by CSPC Pharmaceutical Limited Cooperation (China) has been approved by the SFDA, China, for the treatment of brain tumours and other carcinomas [ ].

Techniques: