#Tumour cells have aberrant activation of mTORC1 that induces an anabolic growth program resulting in nucleotide, protein, and lipid synthesis. Loss of tumour suppressors like p53 or activation of oncogenes like MYC further promotes anabolism through transcriptional regulation of metabolic genes. Metabolism controls signaling through regulating reactive oxygen species (ROS), acetylation, and methylation. PPP, pentose phosphate pathway; G6P, glucose-6-phosphate; 3-PG, 3-phosphoglycerate; ATP, adenosine 5´-triphosphate; mTORC1, mTOR complex 1; α-KG, α-ketoglutarate; RTK, receptor tyrosine kinase.
Tuesday, July 17, 2018
Monday, July 16, 2018
Friday, July 13, 2018
#Sclerostin induced #, and osteolysis in #breast cancer
Expression of sclerostin in human breast cancer cell lines. (A)Expression of sclerostin protein. GAPDH was used as an internal control. Sclerostin protein was extracted from supernatant culture medium after centrifugation and concentration. (B) Quantification of sclerostin are normalizing to GAPDH. (C) Expression of sclerostin mRNA (RT-PCR: 45 cycles of amplification). GAPDH mRNA was used as an internal control. Each bar represents mean ± SEM.
Effect of sclerostin inhibition on cell proliferation, migration and invasion. MTT assays were performed at 1d, 2d, and 3d after treatment with sclerostin antibody (at 0, 1 and 4 µg/ml, respectively) in (A) MCF-7 and (B) MDA-MB-231 cells. (C) Cell migration assays were performed using Transwell system. MDA-MB-231 and MCF-7 cells were treated with sclerostin antibody (at 1 µg/ml and 4 µg/ml, respectively) for 24 h. Migrated cells were stained with crystal violet and (D) migration capability was evaluated by counting migrated cells. (E) Invasion assays were performed and (F) invasiveness was quantified by counting invading cells. NC, non-treated control group; 1 µg/ml, 1 µg/ml sclerostin antibody-treated group; 4 µg/ml, 4 µg/ml sclerostin antibody-treated group. Each bar represents mean ± SEM. *P < 0.05, **P < 0.01. All experiments were performed at least three times with duplication within each individual experiment.
Effect of Scl-Ab on tumor growth in vivo. A breast cancer xenograft model was established using MDA-MB-231 cells (5 × 10A) Tumor size was measured using Vernier caliper once a week until the animals were sacrificed after 40 days of treatment. (B) Tumor weight was measured at the last time point. (C) Kaplan-Meier survival plot of xenografted control, PBS-treated and Sci-Ab treated groups. (D) Sclerostin protein expression and quantification (E) in tumor tissues of the above three groups. NC, non-treated control group; PBS, PBS-treated group; Sci-Ab, sclerostin antibody-treated group (1 μg antibody in 20 μl PBS). Each bar represents mean ± SEM. NS, non-significant.cells in 30 μl PBS) implanted into the femur of female nude mice (7–8 weeks old). (
Analyses of tumor characteristics and serum of xenografted mice. (A) Representative H&E staining and immunohistochemistry for sclerostin (100 × magnification) in tumor sections obtained from different groups. (B) Quantification of osteocalcin (OCN), (C) osteoprotegerin (OPG), and (D) sclerostin in serum of mice from different groups by ELISA. NC, non-treated control group; PBS, PBS-treated group; Scl-Ab, sclerostin antibody-treated group (1 μg antibody in 20 μl PBS). Each bar represents mean ± SEM. **P < 0.01, NS, non-significant.
In vivo micro-CT analysis. (A) Presence of tumor-induced osteolytic lesions detected by micro-CT scans. Representative 3-D reconstruction of micro-CT images of femurs from non-treated control, PBS- and Sci-Ab treated mice. Three-dimensional images reconstructed from micro-CT analysis on the cortical and trabecular bone microarchitecture of whole left femur (B, longitudinal section) and of distal femoral metaphysis (C, cross section) in different groups. On Micro-CT, NC and PBS mice showed increased (D) trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), (F) bone volume/tissue volume ratio (BV/TV), (G) bone mineral density (BMD), and (H) cortical bone volume/tissue volume ratio (cortical BV/TV), whereas decreased (I) trabecular separation (Tb.Sp) and (J) bone surface/bone volume ratio (BS/BV) compared with Scl-Ab treated mice. NC, non-treated control group; PBS, PBS-treated group; Scl-Ab, sclerostin antibody-treated group (1 μg antibody in 20 μl PBS). Each bar represents mean ± SEM. *P < 0.05, **P < 0.01; NS, non-significant.
Monday, July 9, 2018
Mechanisms of lipid-induced cellular injury in NAFLD. ROS are formed through oxidative processes within the cell. In the mitochondria, impaired MRC activity leads to the formation of superoxide anions and hydrogen peroxide. The accumulation of fatty acids in the cytosol increases fatty acid oxidation in peroxisomes and the ER. The initial reaction in peroxisomal β oxidation is catalyzed by acyl-CoA oxidase (AOX) that forms hydrogen peroxide through the donation of electrons to molecular oxygen. Microsomal w oxidation is catalyzed by cytochrome P450 (CYP) enzymes 2E1, 4A10, and 4A14, which form ROS through flavoprotein-mediated donation of electrons to molecular oxygen. PUFAs are extremely susceptible to lipid peroxidation by ROS. By-products of PUFA peroxidation are aldehydes, such as HNE and MDA. These aldehydes are themselves cytotoxic and can freely diffuse into the extracellular space to affect distant cells. ROS and aldehydes induce oxidative stress and cell death via ATP and NAD depletion, DNA and protein damage, and glutathione depletion. Additionally, they induce inflammation through the production of proinflammatory cytokines, leading to neutrophil chemotaxis. Within the extracellular space, HNE and MDA are themselves potent chemoattractants for neutrophils. Finally, ROS and products of lipid peroxidation can lead to fibrosis by activating hepatic stellate cells, which synthesize collagen and perpetuate the inflammatory response.