Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: GBP2 is downregulated and acts as a prognostic marker of TNBC. (A) mRNA levels of GBP2 in breast tumor tissues and normal breast tissues (Normal: n=113; Tumor: n=1,052). (B) GBP2 mRNA levels in four breast cancer subtypes and normal breast tissues (Normal: n=113; Basal: n=190; HER2: n=81; LumA: n=564; LumB: n=217. The data were obtained from The Cancer Genome Atlas database. (C) Kaplan-Meier plot analysis analyzed the association between high/low GBP2 expression and relapse-free survival in human breast cancer and its subtypes. (D) GBP2 mRNA levels assayed by reverse transcription-quantitative PCR. (E) Protein levels of GBP2 examined by western blotting. (F) Representative images of immunohistochemical staining of GBP2 in tumor/normal tissue samples. ** P<0.01 and *** P<0.001. TNBC, triple-negative breast cancer; GBP2, guanylate-binding protein 2; LumA, Luminal A; LumB, Luminal B.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Marker, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Binding Assay

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: GBP2 protein plays a crucial role in suppressing triple-negative breast cancer cells. The expression of GBP-2 was forced by lentiviral transfection, and the transfection efficiency of the gene was confirmed before further experiments. (A and B) Western blot defined the expression level of GBP-2 protein. Relative protein expression of GBP2 is shown in the bar graph. (C) Reverse transcription-quantitative PCR revealed the mRNA level of GBP-2. (D-F) The impact of overexpressing GBP2 on proliferation in MDA-MB-231/436 cells was assessed utilizing the (D) Cell Counting Kit-8 and (E and F) colony formation assays. (G and H) Effect of GBP2 overexpression on the migratory capacity of MDA-MB-231/436 cells analyzed by a wound healing assay. (I and J) The invasive ability of MDA-MB-231/436 cells was evaluated using the Transwell assay to measure the impact of overexpressing GBP2; images of random visual fields were captured at 24 h to record the number of cells per visual field. * P<0.05 and ** P<0.01. GBP2, guanylate-binding protein 2; OE, (GBP2) overexpression group; NC, negative control.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Over Expression, Wound Healing Assay, Transwell Assay, Binding Assay, Negative Control

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: Expression level of GBP2 is influenced by the concentration of paclitaxel. (A) GBP2 protein expression level was detected utilizing WB after 24 h of treatment with various concentrations (0, 5, 10 and 15 nM) of paclitaxel. (B) Data are quantified by bar graphs. (C and D) The NC/PR groups of MDA-MB-231 and MDA-MB-436 cells were exposed to increasing doses of paclitaxel for 24 h, and then the cell viability was detected by Cell Counting Kit-8 assay, separately. The IC 50 value was calculated using SSPS. (E) Demonstration of a concentration-induced method for establishing paclitaxel-resistant MDA-MB-231/436 cell lines (maintained at a final concentration of 50 nM). (F and G) The relative mRNA and (H and I) protein levels of GBP2 were assessed using reverse transcription-quantitative PCR and WB, respectively (n=3). ** P<0.01. GBP2, guanylate-binding protein 2; WB, western blotting; NC, negative control. PR, paclitaxel-resistant.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Expressing, Concentration Assay, Cell Counting, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot, Negative Control

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: GBP2 overexpression improves the sensitivity to paclitaxel in triple-negative breast cancer. GBP2 was overexpressed in drug-resistant MDA-MB-231 cells by adenoviral transfection (PR + OE), and the expression level of GBP2 was measured using reverse transcription-quantitative PCR and western blotting. (A and B) Relative protein and (C) mRNA of GBP2 was significantly increased in the PR + OE group. (D) The cell viability was assessed using the Cell Counting Kit-8 assay after treatment with increasing amounts of paclitaxel for 24 h. (E and F) Colony formation assay was used to detect cell proliferation in each group. (G and H) Transwell assays and (I and J) gap closure assays were utilized to detect the migration and invasion, respectively. * P<0.05 and ** P<0.01. GBP2, guanylate-binding protein 2; PR, paclitaxel-resistant cells; PR + OE, paclitaxel-resistant cells + GBP2-overexpression; NC, negative control.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Over Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Colony Assay, Migration, Binding Assay, Negative Control

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: Overexpression of GBP2 activates autophagy in triple-negative breast cancer. Autophagy was detected in MDA-MB-231 parental cells (NC) and stable GBP2 overexpressing (OE) cells. (A and B) Projection electron microscopy revealed autophagosome-like structures (white arrows), and single-field vesicles were counted. (C and D) Autophagic flux was analyzed using the mRFP-GFP-LC3 construct. (E and F) LC3 and p62 expression levels were analyzed by WB. ATG2 and ATG5 were detected by WB as representatives of ATG family. (G and H) Immunofluorescence colocalization indicated GBP2 and ATG2 protein expression and locations in both cell groups. Pearson's correlation coefficients for GBP2 and ATG2 were calculated using ImageJ software. ** P<0.01. GBP2, guanylate-binding protein 2; WB, western blotting; ATG, autophagy-related gene; OE, GBP2-overexpression group; NC, negative control; ns, no significance.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Over Expression, Electron Microscopy, Construct, Expressing, Immunofluorescence, Software, Binding Assay, Western Blot, Negative Control

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: GBP2 overexpression negatively regulates the PI3K/Akt/mTOR pathway. (A) Western blot analysis was performed on total cell lysates from MDA-MB-231 parental (NC) and stable GBP2 overexpressing (OE) cells to evaluate the major PI3K/Akt/mTOR pathway proteins. (B and C) Change in expression of each key protein and their phosphorylation is quantified as the bar graph (n=3). ** P<0.01. GBP2, guanylate-binding protein 2; OE, GBP2-overexpression group; NC, negative control; p-, phosphorylated; ns, no significance.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Over Expression, Western Blot, Expressing, Binding Assay, Negative Control

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: Enhanced autophagy by GBP2 leads to the reversal of paclitaxel resistance in triple-negative breast cancer cells. GBP2 overexpression or empty adenovirus was transfected into MB231 paclitaxel-resistant cells, and cells were treated with CQ (50 μ mol/l) or CC (7.5 μ mol/ml) or none for 24 h. (A) Ampk and mTOR protein and phosphorylation levels were detected using WB. (B and C) Relative density represents relative phosphorylation protein expression of AMPK and mTOR. (D) Autophagy-related proteins LC3 II/I, P62 and ATG2 were detected using WB. (E-G) The relative protein expression levels were quantified using bar graphs. (H and I) The colony formation assay was utilized to ascertain proliferation of cells in every treatment group. (J) Cell vitality was determined utilizing the Cell Counting Kit-8 assay after treating four groups of cells with increasing concentrations of paclitaxel for 24 h. The IC 50 value was calculated using SSPS, and results from low to high are as follows: 18.0, 31.5, 40.5 and 52.5 nM. ** P<0.01. GBP2, guanylate-binding protein 2; CQ, chloroquine; CC, Compound C; ATG, autophagy-related gene; WB, western blotting; OE, GBP2-overexpression group; p-, phosphorylated.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Over Expression, Transfection, Expressing, Colony Assay, Cell Counting, Binding Assay, Western Blot

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: Overexpression of GBP2 inhibits the growth of triple-negative breast cancer tumors in vivo . (A) BALB/c nude mice were evenly divided into two groups (Con/OE), and GBP2 parent cells and stable overexpressing cells of MDA-MB-231 were subcutaneously implanted separately. The mice were sacrificed 35 days after implantation. (B and C) Weight of body and volume of tumor were recorded every 5 days during feeding. (D-F) After the sacrifice, the tumors were excised and measured. Then tumor slices were fabricated for subsequent experiments. (G and H) EDU staining assay was used to detect tumor cell proliferation in both groups. (I and J) Projection electron microscopy revealed autophagosome-like structures (white arrows). ** P<0.01. GBP2, guanylate-binding protein 2; EDU, 5-ethynyl-2′-deoxyuridine; Con, control group; OE, GBP2-overexpression group.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Over Expression, In Vivo, Staining, Electron Microscopy, Binding Assay

Journal: International Journal of Oncology

Article Title: GBP2 enhances paclitaxel sensitivity in triple-negative breast cancer by promoting autophagy in combination with ATG2 and inhibiting the PI3K/AKT/mTOR pathway

doi: 10.3892/ijo.2024.5622

Figure Lengend Snippet: GBP2 overexpression is revealed to activate autophagy in triple-negative breast cancer in vivo . The isolated tumor sections were further examined using immunofluorescence. (A) Immunofluorescence staining confirmed the overexpression of GBP2 in the tumor cells of the OE group. (B-D) LC3, p62 and ATG2 expression levels were determined using immunofluorescence in tumors from both groups. (E) Relative expression of the aforementioned proteins in the two groups of tumors was quantified using bar graphs. ** P<0.01. GBP2, guanylate-binding protein 2; ATG, autophagy-related gene; OE, GBP2-overexpression group; Con, control group.

Article Snippet: A total of 1.5 μ g of pBI-GBP2-EGFP vector (cat. no. 6154-1; BD Biosciences), which expresses GBP2 with the C-terminal EGFP tag, and 2 μ g of pIRESpuro plasmid (cat. no. KL101-994; Shanghai kanglang biological technology Co., Ltd.) were co-transfected into 293T cells (Shanghai GenePharma Co., Ltd.) to generate the lentivirus.

Techniques: Over Expression, In Vivo, Isolation, Immunofluorescence, Staining, Expressing, Binding Assay