Journal: Acta Neuropathologica Communications

Article Title: Inhibition of mitochondrial respiration prevents BRAF -mutant melanoma brain metastasis

doi: 10.1186/s40478-019-0712-8

Figure Lengend Snippet: In vitro drug screening. a Heatmap of three days H1_DL2 monolayer proliferation assays (left panel) and representative transmission (Trans) and 4′,6-Diamidino-2-Phenylindole (DAPI) images (right panel). b Heatmap of ten days H1_DL2 tumorsphere assays (left panel) and representative differential interference contrast (DIC) and green fluorescent protein (GFP) fluorescence microscopy images (right panel). a-b Right image panels show examples of β-sitosterol 218.7 μM (top), β-sitosterol 2.7 μM (middle) and control (bottom). c Half maximal inhibitory concentration (IC 50 ) values for the three most potent compounds across different cell lines and assays. (a–c) Mean; n = 6 per cell line per drug per drug concentration. See Additional file : Figure S3a–c for more details on this experiment

Article Snippet: Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C).

Techniques: In Vitro, Transmission Assay, Fluorescence, Microscopy, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: Inhibition of mitochondrial respiration prevents BRAF -mutant melanoma brain metastasis

doi: 10.1186/s40478-019-0712-8

Figure Lengend Snippet: In vivo drug screening. a Experimental overview: Intracardiac injections of 5 × 10 H1_DL2 cells in NOD/SCID mice were followed by MRI-based quantification of nanoparticle-labeled melanoma cells in the mouse brains after 24 h for group homogenization (Additional file : Figure S4a). Treatment started after one week, and test groups received 0.2 mL i.p. injections of 40 mg/kg thiostrepton every second day ( n = 5), 10 mg/kg memantine daily (n = 5), 5 mg/kg β-sitosterol daily ( n = 4) or vehicle (0.5% DMSO; n = 5). See Additional file : Figure S4 for more details. b Development of brain metastases visualized by MRI (T1-weighted images with contrast) and BLI at five, six and seven weeks. Scale bar MRIs, 0.25 cm. c Number of brain metastases at T1-weighted MRI with contrast (Student’s t -test). d Kaplan-Meier survival plot (Mantel-Cox log-rank test). There was no significant difference between vehicle- and memantine-treated mice. * P < 0.05; ** P < 0.01. All values are given as the mean ± s.e.m

Article Snippet: Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C).

Techniques: In Vivo, Labeling, Homogenization

Journal: Acta Neuropathologica Communications

Article Title: Inhibition of mitochondrial respiration prevents BRAF -mutant melanoma brain metastasis

doi: 10.1186/s40478-019-0712-8

Figure Lengend Snippet: In vivo validation of β-sitosterol pre-treatment. a Experimental overview: Treatment started one week prior to intracardiac injections of 5 × 10 5 H1_DL2 cells in NOD/SCID mice. Quantification of tumor cell exposure in the mouse brains was carried out 24 h after injections for group homogenization (Additional file : Figure S5a). Test groups received daily i.p. injections of 0.1 mL vehicle (olive oil; n = 10) or β-sitosterol diluted in olive oil (5 mg/kg; n = 9). Vehicle treatment was continued until euthanization and β-sitosterol was given for ten weeks. See Additional file : Figure S5 for more details on this experiment. b Development of brain metastases assessed by MRI at four, six, seven and eight weeks. Scale bar MRIs (T1-weighted images with contrast), = 0.25 cm. c Number of brain metastases assessed by T1-weighted MRI with contrast (Student’s t -test). The mean number of brain metastases in the vehicle group decreased slightly from seven to eight weeks as four mice with the greatest number of brain metastases were sacrificed between these observation points. d Brain BLI at seven and eight weeks (total photon count = dorsal + ventral region of interest (ROI); Student’s t -test). e Kaplan-Meier survival plot (Mantel-Cox log-rank test). The experiment was terminated at 100 days, and three mice in the β-sitosterol group were still alive and healthy. * P < 0.05; ** P < 0.01; **** P < 0.0001. All values are given as the mean ± s.e.m

Article Snippet: Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C).

Techniques: In Vivo, Homogenization

Journal: Acta Neuropathologica Communications

Article Title: Inhibition of mitochondrial respiration prevents BRAF -mutant melanoma brain metastasis

doi: 10.1186/s40478-019-0712-8

Figure Lengend Snippet: β-sitosterol reduces mitochondrial respiration through complex I inhibition. a-b Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured to assess rates of mitochondrial respiration and glycolysis, respectively, in H1_DL2 cells treated with 50 μM β-sitosterol or 0.05% DMSO for 24 h (both: n = 4). a Basal respiration was determined, followed by sequential additions of oligomycin (3 μM) to assess respiration due to proton leak, carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 1.5 μM) to measure respiratory capacity, rotenone (1 μM) to assess Complex I (CI) independent respiration and antimycin A (AMA; 1 μM) to determine background OCR. b Glucose (10 mM) was provided to determine basal glycolysis, followed by sequential additions of oligomycin (3 mM) to obtain glycolytic capacity, CCCP (1.5 μM) to evaluate the influence of uncoupling and 2-deoxyglucose (2-DG; 100 mM) to measure the non-glycolytic background. c High-resolution respirometry in H1_DL2 cells to detect direct effects of β-sitosterol. First, the maximal CI + CII driven respiratory capacity was measured in the presence of digitonin (8.1 μM), malate (2 mM), pyruvate (1 mM), succinate (10 mM) and carbonylcyanide-4-(trifluoromethoxy)-phenylhydraqone (FCCP, 0.18 μM). The respiratory rate was then measured after adding β-sitosterol (50 μM) or DMSO (0.05%), followed by rotenone (0.5 μM) to inhibit CI, and AMA (2.3 μM) to determine residual oxygen consumption. The experiment was repeated 3 times. a - c Student’s t -test: n.s. = not significant, P ≥ 0.05, **** P < 0.0001. Values are given as the mean ± s.d

Article Snippet: Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C).

Techniques: Inhibition

Journal: Acta Neuropathologica Communications

Article Title: Inhibition of mitochondrial respiration prevents BRAF -mutant melanoma brain metastasis

doi: 10.1186/s40478-019-0712-8

Figure Lengend Snippet: β-sitosterol increases ROS production and apoptosis. a ROS content (CM-H 2 DCFDA probe; mean fluorescence intensity (MFI); n = 2 with triplicates). b Flow cytometric apoptosis assay (n = 3) showing a strong induction of apoptosis following ß-sitosterol treatment. c Western blot of pro-caspase-3, cleaved caspase-3 and GAPDH in H1_DL2 cells exposed to DMSO (0.05%) or β-sitosterol (50 μM) for 2, 24 or 24 h, respectively. Student’s t -test: *** P < 0.001, **** P < 0.0001. Values are given as the mean ± s.e.m

Article Snippet: Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C).

Techniques: Fluorescence, Apoptosis Assay, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Inhibition of mitochondrial respiration prevents BRAF -mutant melanoma brain metastasis

doi: 10.1186/s40478-019-0712-8

Figure Lengend Snippet: Mitochondrial complex I inhibition prevents BRAFi resistance. a . Western blot of PGC1α and GAPDH in H1 cells treated with DMSO (0.05%) or β-sitosterol (12.5, 25 or 50 μM) for 24 h (n = 3). b . MitoTracker Red mean fluorescence intensity in H1 cells treated with DMSO (0.05%) or vemurafenib (1.5 μM) for 72 h (n = 3). c Colony formation assay (crystal violet staining) of H1 cells treated with DMSO (0.05%) for 1 week, or vemurafenib (1.5 μM), β-sitosterol (50 μM), or vemurafenib + β-sitosterol for 3 weeks (n = 3). d Western blot of NDUFA8 and β-actin in H1_shCtr and H1_shNDUFA8 cells (NDUFA8 is required for assembly of a functional complex I). e Colony formation assay of H1 cells treated with DMSO (0.05%) for one week or vemurafenib (1.5 μM) for three weeks, and H1_shNDUFA8 cells alone or treated with vemurafenib (1.5 μM) for three weeks (n = 3). f Half maximal inhibitory concentration (IC 50 ) values for β-sitosterol, PLX4720 and the combination thereof in Melmet 1, Melmet 5 and A375 cell lines (n = 3). g Subcutaneous tumor volume (width 2 × length)/2) in mice injected with 1 × 10 6 Melmet 5 cells. Mice were from two weeks onwards given daily i.p. injections of 0.1 mL vehicle (olive oil), 20 mg/kg β-sitosterol in olive oil, 25 mg/kg PLX4720 in 0.05% DMSO, or a combination of the two latter ( n = 8 in each group). Representative images of tumors at 44 days are shown in the right panel. Student’s t -test: * P < 0.05. Values are given as the mean ± s.e.m

Article Snippet: Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C).

Techniques: Inhibition, Western Blot, Fluorescence, Colony Assay, Staining, Functional Assay, Concentration Assay, Injection

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: Molecular clustering diagram. The diagram shows three clusters of small molecules: the first cluster is green representing Sitogluside, the second cluster is brown representing Kaempferol, and the third cluster is gray representing Nuciferin.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: The Venn diagram illustrates the shared intersection genes between lotus leaf small molecules and obesity disease. (A) The number of shared intersection genes between Sitogluside and obesity disease is 13. (B) The number of shared genes between Kaempferol and obesity disease is 48. (C) The number of shared intersection genes between Nuciferin and obesity disease is 39.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: The diagram illustrates protein interactions involved in lotus leaf treatment of obesity. (A) Top 10 gene interactions of Sitogluside. (B) Top 10 gene interactions of Kaempferol. (C) Top 10 gene interactions of Nuciferin.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: The results presented are based on the GO (A) and KEGG (B) pathway enrichment analyses of intersection genes between Sitogluside and obesity.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: Compound_name and docking energy results.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: Docking results of PPARG with two active compounds from lotus Leaf. (A) Interaction between sitogluside and PPARG. (B) Interaction between cycloartenol and PPARG.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: Secondary structure analysis of the protein in three systems. (A) Apo (B) Sitogluside (C) Cycloartenol.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: PCA analysis of the three systems. (A) Apo. (B) Sitogluside (C) Cycloartenol. Covariance matrix analysis of three systems. (D) Apo. (E) Sitogluside (F) Cycloartenol.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: The result of MM-PBSA.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: MM-PBSA energy contribution and hydrogen bonds. (A) Sitogluside (B) Cycloartenol.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Investigating the anti-obesity potential of Nelumbo nucifera leaf bioactive compounds through machine learning and computational biology methods

doi: 10.3389/fphar.2024.1500865

Figure Lengend Snippet: Effects of Sitogluside on Lipid Accumulation and Triglyceride Levels in 3T3-L1 Cells. (A–C) Representative microscopy images of 3T3-L1 cells stained with Oil Red O showing lipid accumulation at Sitogluside concentrations of 0 µM (A) , 5 µM (B) , and 10 µM (C) . (D) Graph illustrating the percentage of lipid accumulation in 3T3-L1 cells at different concentrations of Sitogluside. A clear dose-dependent decrease in lipid accumulation is observed. (E) Optical density (OD) at 490 nm of extracted dye from Oil Red O staining, demonstrating decreased lipid content with increasing concentrations of Sitogluside. (F) Triglyceride (TG) concentration measured in µmol/L, showing a significant reduction in triglyceride levels as the concentration of Sitogluside increases.

Article Snippet: Sitogluside was obtained from TargetMol.

Techniques: Microscopy, Staining, Concentration Assay

Journal: Bioengineered

Article Title: An extraction from Trametes robiniophila Murr. ( Huaier ) inhibits non-small cell lung cancer proliferation via targeting to epidermal growth factor receptor

doi: 10.1080/21655979.2022.2066757

Figure Lengend Snippet: Virtual verification of Huaier targets by molecular docking. (a) Maestro 2D interactions between EGFR and gefitinib, adenosine, daucosterol, ergosterol, ergosta-7,22-dien-3β-ol and 3β-hydroxystigmast-5,22-dien-7-one. Residues in green spheres are hydrophobic, blue spheres are polar, red spheres are negatively charged, purple spheres are charged and light yellow spheres are glycine. The purple arrows and their directions represent hydrogen bonds between the ligand and the protein. The green line represents the π-π stacking arrangement seen between the aromatic core. (b) We conducted kinase activity assays to validate Huaier ’s inhibition on EGFR kinase activity. Staurosporine, an effective PKC inhibitor, was chosen as positive control. (c) Cells were conducted by CETSAs, and the protein expression of EGFR was detected by western blot analysis. The line chart showed the relative expression level of EGFR.

Article Snippet: Gefitinib and Daucosterol were both purchased from MCE (HY-50895; HY-N0410).

Techniques: Activity Assay, Inhibition, Positive Control, Expressing, Western Blot