Journal: Oxidative Medicine and Cellular Longevity

Article Title: HSP90AB1 as the Druggable Target of Maggot Extract Reverses Cisplatin Resistance in Ovarian Cancer

doi: 10.1155/2023/9335440

Figure Lengend Snippet: ME enhances the sensitivity of A2780/CDDP and SKOV3/CDDP cells to cisplatin (Cis). Cells were treated with different concentrations of cisplatin with and without ME (6 mg/ml) for 48 h. (a) The viability of A2780 and A2780/CDDP cells was assayed using CCK8 kits. (b) The viability of SKOV3 and SKOV3/CDDP cells was assayed. SKOV3/CDDP cells were infected with lentiviral particles to express luciferase, and then cells were treated with cisplatin (3.2 μ g/ml) and/or ME (4 mg/ml or 6 mg/ml) for 48 h followed by 150 μ g/ml D-luciferin for 10 min. (c) The luciferase-positive SKOV3/CDDP cells were analyzed using Living Image software and a GloMax® 96 Microplate Luminometer. The quantification of luciferase-positive cells is shown on the right. ∗ p ≤ 0.05 vs. blank; # p ≤ 0.05 vs. cisplatin; (d) A2780/CDDP and SKOV3/CDDP cells were double stained with Annexin V-FITC and PI then analyzed using flow cytometry, and the quantitative analysis was located in the right. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. (e) Levels of p-p53 in A2780/DDP cells were measured by immunofluorescence staining (400x magnification). The panel under the staining shows the quantitative analysis of p-p53 in A2780/CDDP cells. The nuclei are stained with DAPI. ∗ p ≤ 0.05 vs. blank; # p ≤ 0.05 vs. cisplatin. (f) Representative images of the wound healing assays in A2780/CDDP and SKOV3/CDDP cells are shown, and the panels under the images show the wound closure rate. ∗ p ≤ 0.05 vs. 0 h; # p ≤ 0.05 vs. blank; ns means no significance vs. 0 h. (g) The transwell assay was used to analyze the invasion of A2780 and A2780/CDDP cells. The invasive cell number was quantified using ImageJ and located under the images. ∗ p ≤ 0.05. Three independent experiments were performed with similar results. Data are shown as the mean ± SEM.

Article Snippet: The human ovarian cancer cell line A2780 (RRID: CVCL_0134) was purchased from Shanghai Pituo Biological Technology Co., Ltd. (China), and the cell line SKOV3 (RRID: CVCL_0532) was purchased from Solarbio Science & Technology Co., Ltd. (China).

Techniques: Infection, Luciferase, Software, Staining, Flow Cytometry, Immunofluorescence, Transwell Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: HSP90AB1 as the Druggable Target of Maggot Extract Reverses Cisplatin Resistance in Ovarian Cancer

doi: 10.1155/2023/9335440

Figure Lengend Snippet: Enrichment analysis of DEGs in A2780/CDDP cells based on high-throughput RNA sequencing. The total RNA from A2780 and A2780/DDP cells was extracted using RNA Miniprep kit reagents. Next-generation sequencing analysis was performed on the BGISEQ-500 platform by BGI Genomic Services. (a) Volcano plot showing significantly upregulated genes (red dots) and downregulated genes (blue dots). (b) A2780 vs. A2780/CDDP Gene Ontology (GO) analysis on a cellular component of DEGs. (c) A2780 vs. A2780/CDDP KEGG pathway enrichment analysis of DEGs. (d) The heat map shows the relative transcript levels of the DEGs in A2780 and A2780/CDDP cells. (e) The protein–protein interaction network shows the upregulated DEGs from A2780/CDDP cells compared with A2780 cells. (f) qRT-PCR analyses of the mRNA levels of MYC in A2780 cells and A2780/CDDP cells. (g) qRT-PCR analyses of the mRNA levels of HSP90AB1 in A2780 cells and A2780/CDDP cells. Three independent experiments were performed with similar results. Data are shown as the mean ± SEM. ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001.

Article Snippet: The human ovarian cancer cell line A2780 (RRID: CVCL_0134) was purchased from Shanghai Pituo Biological Technology Co., Ltd. (China), and the cell line SKOV3 (RRID: CVCL_0532) was purchased from Solarbio Science & Technology Co., Ltd. (China).

Techniques: High Throughput Screening Assay, RNA Sequencing, Next-Generation Sequencing, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: HSP90AB1 as the Druggable Target of Maggot Extract Reverses Cisplatin Resistance in Ovarian Cancer

doi: 10.1155/2023/9335440

Figure Lengend Snippet: ME induces cisplatin to promote apoptosis by suppressing the expression of HSP90AB1/IGF1R in cisplatin-resistant ovarian cancer cells. (a) The expression of HSP90AB1, IGF1R, and IGFBP2 in A2780 cells and A2780/CDDP cells was assessed by western blot. (b) The lower panel shows the quantitative analysis. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. (c) The expression of HSP90AB1, IGF1R, and IGFBP2 in SKOV3 cells and SKOV3/CDDP cells was assessed by western blot. (d) The lower panel shows the quantitative analysis. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01. The GSH level in A2780 cells (e) and SKOV3 cells (f) was analyzed using a reduced GSH assay kit. ∗ p ≤ 0.05, ns means no significance. Relative mRNA expression of MYC (g), HSP90AB1 (h), and IGF1R (i) in A2780/CDDP cells was determined by real-time PCR. ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001, ns means no significance. A2780/CDDP and SKOV3/CDDP cells were treated with cisplatin (3.2 μ g/ml) and ME (6 mg/ml) for 48 h. The protein was extracted for further analysis. (j–m) The protein expression of HSP90AB1, IGF1R, MYC, PTEN, p-H2AX, and BCL2 in A2780/CDDP cells (j) and SKOV3/CDDP cells (l) was measured by western blot. The lower panel shows the quantitative analysis of the western blot in A2780/CDDP cells (K) and SKOV3/CDDP cells (m). ∗ p ≤ 0.05, ns means no significance. Drug-resistant cells were treated with cisplatin, ME, or both. The amount of GSH in A2780/CDDP cells (n) and SKOV3/CDDP cells (o) was analyzed using a reduced GSH assay kit. ∗ p ≤ 0.05, ns means no significance. Three independent experiments were performed with similar results. Data are shown as the mean ± SEM.

Article Snippet: The human ovarian cancer cell line A2780 (RRID: CVCL_0134) was purchased from Shanghai Pituo Biological Technology Co., Ltd. (China), and the cell line SKOV3 (RRID: CVCL_0532) was purchased from Solarbio Science & Technology Co., Ltd. (China).

Techniques: Expressing, Western Blot, GSH Assay, Real-time Polymerase Chain Reaction

Journal: Oxidative Medicine and Cellular Longevity

Article Title: HSP90AB1 as the Druggable Target of Maggot Extract Reverses Cisplatin Resistance in Ovarian Cancer

doi: 10.1155/2023/9335440

Figure Lengend Snippet: Inhibition of HSP90 ATPase activity with geldanamycin promotes apoptosis and suppresses migration in cisplatin-resistant ovarian cancer cells. A2780/CDDP cells and SKOV3/CDDP cells were pretreated with 5 μ M geldanamycin (In-HSP) for 1 h and then treated with cisplatin (3.2 μ g/ml) and ME (6 mg/ml) for 72 h. (a) A2780/CDDP cells and SKOV3/CDDP cells were double stained with Annexin V-FITC and PI and then analyzed by flow cytometry, and the quantitative analysis was located on the right. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. (b, c) A2780/CDDP cells and SKOV3/CDDP cells expressing luciferase were pretreated with 150 μ g/ml D-luciferin for 10 min. The luciferase-positive A2780/CDDP cells (b) and SKOV3/CDDP cells (c) were analyzed using Living Image software and a GloMax® 96 Microplate Luminometer. The quantification of luciferase-positive cells is shown on the right. ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. Representative images of the wound healing assays of A2780/CDDP cells (d) and SKOV3/CDDP cells (e) are shown, and the quantification of the wound closure rate is on the right. ∗ p ≤ 0.05 vs. 0 h; ns mean no significance vs. 0 h; ## p ≤ 0.01. (f) The expression of HSP90AB1, IGF1R, p-H2AX, p-p53, and BAX in A2780/CDDP cells was analyzed by western blot. (g) The panel on the right shows the quantitative analysis. ∗ p ≤ 0.05. (h) The expression of HSP90AB1, IGF1R, p-H2AX, p-p53, and BCL2 in SKOV3/CDDP cells was measured by western blot. (i) The panel on the right shows the quantitative analysis. ∗ p ≤ 0.05. (j–m) The coimmunoprecipitation assay. SKOV3/CDDP cells were treated with geldanamycin for 24 h. The expression levels of HSP90AB1 and IGF1R were measured (j), and the panel on the right shows the quantitative analysis (k). ∗ p ≤ 0.05. The same cell lysates were immunoprecipitated with IGF1R, HSP90AB1, and isoform-matched immunoglobulin (IgG). (l) Western blot assays of SKOV3/CDDP cells using site-specific antibodies against HSP90AB1 and IGF1R, and the panel on the right shows the quantitative analysis (m). ∗ p ≤ 0.05. Three independent experiments were performed with similar results. Data are shown as the mean ± SEM.

Article Snippet: The human ovarian cancer cell line A2780 (RRID: CVCL_0134) was purchased from Shanghai Pituo Biological Technology Co., Ltd. (China), and the cell line SKOV3 (RRID: CVCL_0532) was purchased from Solarbio Science & Technology Co., Ltd. (China).

Techniques: Inhibition, Activity Assay, Migration, Staining, Flow Cytometry, Expressing, Luciferase, Software, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation