Journal: Biomolecules & Therapeutics

Article Title: 6-Gingerol Induced Apoptosis and Cell Cycle Arrest in Glioma Cells via MnSOD and ERK Phosphorylation Modulation

doi: 10.4062/biomolther.2024.084

Figure Lengend Snippet: 6-gingerol exhibits BBB permeability and cytotoxic potential in M059K and U251 cells. (A) Simulations were conducted using Support Vector Machine (SVM) algorithms to predict the potential BBB permeability of 6-gingerol and curcumin. (B) Cell viability was assessed following incubation with varying concentrations of 6-gingerol for 24 to 72 h using the CCK-8 assay. (C) M059K and U251 GBM cells were incubated with different concentrations of 6-gingerol for 16 days, and their ability to form colonies was assessed. The lower panel presents quantitative analyses of colony formation. (D) Concentration-dependent morphological changes, such as cell detachment and cell death, were observed in GBM cells treated with 6-gingerol for 48 h. Light microscopy images (magnification ×100). The scale bar represents 100 mm. The results represent the mean ± SD from three independent experiments; **** p <0.001, *** p <0.005, ** p <0.01, * p <0.05 compared to the respective control group.

Article Snippet: Human GBM cell lines M059K and U251, sourced from the American Type Culture Collection (ATCC), Manassas, VA, USA, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Permeability, Plasmid Preparation, Incubation, CCK-8 Assay, Concentration Assay, Light Microscopy, Control

Journal: Biomolecules & Therapeutics

Article Title: 6-Gingerol Induced Apoptosis and Cell Cycle Arrest in Glioma Cells via MnSOD and ERK Phosphorylation Modulation

doi: 10.4062/biomolther.2024.084

Figure Lengend Snippet: Cell cycle modulation and protein expression changes in response to 6-gingerol treatment. (A) Treatment of M059K and U251 cells with varying concentrations of 6-gingerol for 48 h, followed by flow cytometry analysis to assess the cell cycle distribution. The graph illustrates the proportion of cells in each stage of the cell cycle. (B) Analysis of protein expression in M059K and U251 cells after 48 h of treatment with 6-gingerol. western blot analysis was conducted for Cyclin A, Cyclin B1, Cyclin D1, Cyclin D3, p21, E2F1 and E2F3, using cell lysates from M059K and U251 cells. The lower panel presents a quantitative analysis of western blot data, illustrating the observed changes in protein expression, with GAPDH used as the internal control. The results represent the mean ± SD from three independent experiments; **** p <0.001,*** p <0.005, ** p <0.01, * p <0.05 compared to the respective control group.

Article Snippet: Human GBM cell lines M059K and U251, sourced from the American Type Culture Collection (ATCC), Manassas, VA, USA, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Flow Cytometry, Western Blot, Control

Journal: Biomolecules & Therapeutics

Article Title: 6-Gingerol Induced Apoptosis and Cell Cycle Arrest in Glioma Cells via MnSOD and ERK Phosphorylation Modulation

doi: 10.4062/biomolther.2024.084

Figure Lengend Snippet: Apoptosis induction and protein expression changes in response to 6-gingerol exposure. (A) After a 48 h exposure to 6-gingerol, apoptosis in M059K and U251 cell lines was assessed using cytofluorimetry. The proportion of apoptotic cells was determined through Annexin V-FITC and propidium iodide staining. The right panel illustrates the percentages of cells in early apoptosis (Annexin V+/PI-) and late apoptosis (Annexin V+/PI+), calculated as Annexin V-positive cells for statistical analysis. (B, C) The expression of pro- and anti-apoptotic proteins in M059K and U251 cells was analyzed following a 48 h exposure to 6-gingerol. This analysis included anti-Bax and Bcl-2, Cleaved Caspase-3, Cleaved Caspase-9, and PARP/cleaved PARP. Western blot analysis of lysates from M059K and U251 cells was conducted. GAPDH served as the internal control. The right panel provides a quantitative assessment of western blot data, indicating observed changes in protein expression. All results are presented as mean ± SD from three independent experiments; **** p <0.001, *** p <0.005, ** p <0.01, * p <0.05 compared to the respective control.

Article Snippet: Human GBM cell lines M059K and U251, sourced from the American Type Culture Collection (ATCC), Manassas, VA, USA, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Staining, Western Blot, Control

Journal: Biomolecules & Therapeutics

Article Title: 6-Gingerol Induced Apoptosis and Cell Cycle Arrest in Glioma Cells via MnSOD and ERK Phosphorylation Modulation

doi: 10.4062/biomolther.2024.084

Figure Lengend Snippet: Mito-TEMPO pretreatment partially reverses 6-gingerol-induced mtROS accumulation, ΔΨm reduction, and apoptosis. (A) The impact of 6-gingerol treatment on the reduction of ΔΨm in M059K and U251 cells after 48 h was evaluated using cell-permeable cationic TMRM and flow cytometry. The right panel presents the percentages of M059K and U251 cells with disrupted ΔΨm, which increased with escalating concentrations of 6-gingerol. (B) mtROS levels were assessed by DHE staining and analyzed via flow cytometry in M059K and U251 cells following a 48 h treatment with various concentrations of 6-gingerol. The right panel illustrates the quantification of mtROS accumulation induced by 6-gingerol. (C) Cell lysates were subjected to western blot analysis using an anti-SOD2 antibody, with GAPDH as the internal control. The right panel displays quantified SOD2 protein levels normalized to GAPDH. (D, E) Flow cytometry histograms depict alterations in ΔΨm in M059K and U251 cells. Following a 2 h incubation with 5 μM Mito-TEMPO, cells were treated with or without 100 μM 6-gingerol for 48 h. Results show a reduction in intact ΔΨm alongside an increase in mtROS levels compared to untreated cells. (F) Cleaved Caspase-3 and -9 protein expression were analyzed in M059K and U251 cells. The right panel provides a quantitative assessment of western blot data, indicating observed changes in protein expression. All data are presented as mean ± SD from three independent experiments; **** p <0.001, *** p <0.005, ** p <0.01, * p <0.05 compared to the respective control.

Article Snippet: Human GBM cell lines M059K and U251, sourced from the American Type Culture Collection (ATCC), Manassas, VA, USA, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Flow Cytometry, Staining, Western Blot, Control, Incubation, Expressing

Journal: Biomolecules & Therapeutics

Article Title: 6-Gingerol Induced Apoptosis and Cell Cycle Arrest in Glioma Cells via MnSOD and ERK Phosphorylation Modulation

doi: 10.4062/biomolther.2024.084

Figure Lengend Snippet: 6-gingerol triggers apoptosis and suppresses cellular proliferation through ERK signaling pathway activation. (A) M059K and U251 cells were exposed to varying concentrations of 6-gingerol for 48 h. Subsequently, cells were harvested, and the levels of ERK, p-ERK, JNK, p-JNK, p38, and p-p38 proteins were analyzed through western blotting. (B) Another set of cells was treated with 100 mM 6-gingerol or 10 mM U0126 for 48 h, alongside their respective control conditions. Protein expression levels of ERK, p-ERK, p21, Cyclin D1, and E2F1, which are related to cell cycle regulation, were examined. (C) In both M059K and U251 cells, protein levels of SOD2 and apoptosis-related markers, including Cleaved Caspase-3 and -9, were assessed. GAPDH was used as the internal control. The right panel provides quantitative measurements derived from western blot data, illustrating observed changes in protein expression. All presented data represent the mean ± standard deviation obtained from three independent experiments; **** p <0.001, *** p <0.005, ** p <0.01, * p <0.05 compared to their respective control conditions.

Article Snippet: Human GBM cell lines M059K and U251, sourced from the American Type Culture Collection (ATCC), Manassas, VA, USA, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Activation Assay, Western Blot, Control, Expressing, Derivative Assay, Standard Deviation

Journal: Biomolecules & Therapeutics

Article Title: 6-Gingerol Induced Apoptosis and Cell Cycle Arrest in Glioma Cells via MnSOD and ERK Phosphorylation Modulation

doi: 10.4062/biomolther.2024.084

Figure Lengend Snippet: 6-gingerol disrupts the EGFR/RAS/RAF pathway, leading to the inhibition of MEK/ERK expression. (A) M059K and U251 cells were exposed to varying concentrations of 6-gingerol for 48 h. Subsequently, the cells were harvested, and western blotting was conducted to assess the protein levels of EGFR, p-EGFR, RAS, RAF, and p- RAF. (B) Western blot analysis of EGFR, p-EGFR, ERK, and p-ERK in cell lysates from cells exposed to EGF (100 ng/mL) or 6-gingerol for different durations (0, 15, 30, 45, and 60 min). GAPDH served as the internal control. The right panel provides quantitative measurements derived from western blot data, indicating observed changes in protein expression. All data presented are represented as the mean ± standard deviation from three independent experiments; **** p <0.001, *** p <0.005, ** p <0.01, * p <0.05 compared to their respective control conditions.

Article Snippet: Human GBM cell lines M059K and U251, sourced from the American Type Culture Collection (ATCC), Manassas, VA, USA, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Expressing, Western Blot, Control, Derivative Assay, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GDF15 Drives Glioblastoma Radioresistance by Inhibiting Ferroptosis and Remodeling the Immune Microenvironment

doi: 10.7150/ijbs.115721

Figure Lengend Snippet: GDF15 is upregulated in recurrent GBM and positively correlated with radioresistance. (A) Volcano showing differential gene expression between M059K and M059J. Genes with significantly upregulated expression in M059K are shown in red, while those with significantly downregulated expression are shown in blue. Genes with no significant change in expression are represented in gray. (B) Western blot analysis revealing higher protein levels of GDF15 in the radioresistant U251 cell line. The intensity of the bands was quantified using ImageJ, and the protein expression was normalized to Tubulin. Statistical significance is indicated by *p < 0.05. (C) Transcriptome analysis from public datasets showing upregulated expression of GDF15 in the radioresistant glioma cell line compared to the parental cell line. Data were obtained from GSE207002 , GSE206917 , GSE274090 . (D) Boxplot showing the Radiation Sensitivity Index (RSI) for high versus low expression groups of GDF15 in GBM patients. The RSI was calculated based on ten gene sets, with higher RSI values indicating lower radiation sensitivity. (E) CGGA325 datasets showing GDF15 expression in primary and recurrent GBM tissues (N=291, unit=log10Counts). (F) Representative IHC images of GDF15 expression in primary and recurrent GBM tissues (N=3). Scale bar: 250 μm. IOD/Area quantification was performed by calculating the total optical density (IOD) within the defined ROI divided by the area of the ROI (Area). Higher IOD values indicate stronger protein expression. Data were represented as mean ± SEM. ns, not signifcant; *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Glioblastoma cell lines M059K and M059J were obtained from American Type Culture Collection (ATCC, Cat# CRL-2366 and CRL-2365).

Techniques: Gene Expression, Expressing, Western Blot