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Mutations in isocitrate dehydrogenase (IDH) are frequently found in low-grade gliomas, secondary glioblastoma, chondrosarcoma, acute myeloid leukemias, and intrahepatic cholangiocarcinoma. However, the molecular mechanisms of how IDH2 mutations induce carcinogenesis remain unclear. Using overlapping PCR, transfection, immunoblotting, immunoprecipitation, measurements of enzyme activity, glucose, lactic acid, ATP, and reactive oxygen species (ROS), cell viability, protein degradation assays post-inhibition of the 26S proteasome (bortezomib) or HSP90 (17-AAG), and a homology model, we demonstrated that the properties of ten cancer-associated IDH2 variants (R140G/Q/W and R172S/K/M/W/G/C/P) arising from point mutations are closely related to their structure and stability. Compared with wild-type IDH2, the R172 and R140 point mutations resulted in a decrease in IDH2 activity, ROS, and lactate levels and an increase in glucose and ATP levels under normal and hypoxic conditions, indicating that mutant IDH2 increases cell dependency on mitochondrial oxidative phosphorylation, and reduces glycolysis under hypoxia. Overexpression of most of IDH2 point mutants showed anti-proliferative effects in the 293T and BV2 cell lines by inhibition of PI3K/AKT signaling and cyclin D1 expression and/or induced the expression of TNF-α and IL-6. Furthermore, bortezomib treatment resulted in dramatic degradation of IDH2 mutants, including R140G, R140Q, R140W, R172S and R172K, whereas it had little impact on the expression of WT and other mutants (R172M, R172W, R172G, R172C and R172P). In addition, targeting HSP90 minimally affected the expression of mutated IDH2 due to a lack of interaction between HSP90 and IDH2. The homology model further revealed that changes in conformation and IDH2 protein stability appeared to be associated with these point mutations. Taken together, our findings provide information important for understanding the molecular mechanisms of IDH2 mutations in tumors. [ABSTRACT FROM AUTHOR]

Introduction: The leaves of Morus alba L is a traditional Chinese medicine widely applied in lung diseases. Moracin N (MAN), a secondary metabolite extracted form the leaves of Morus alba L, is a potent anticancer agent. But its molecular mechanism remains unveiled. Objective: In this study, we aimed to examine the effect of MAN on human lung cancer and reveal the underlying molecular mechanism. Methods: MTT assay was conducted to measure cell viability. Annexin V-FITC/PI staining was used to detect cell apoptosis. Confocal microscope was performed to determine the formation of autophagosomes and autolysosomes. Flow cytometry was performed to quantify cell death. Western blotting was used to determine the related-signaling pathway. Results: In the present study, we demonstrated for the first time that MAN inhibitd cell proliferation and induced cell apoptosis in human non-small-cell lung carcinoma (NSCLC) cells. We found that MAN treatment dysregulated mitochondrial function and led to mitochondrial apoptosis in A549 and PC9 cells. Meanwhile, MAN enhanced autophagy flux by the increase of autophagosome formation, the fusion of autophagsomes and lysosomes and lysosomal function. Moreover, mTOR signaling pathway, a classical pathway regualting autophagy, was inhibited by MAN in a time- and dose-dependent mannner, resulting in autophagy induction. Interestingly, autophagy inhibition by CQ or Atg5 knockdown attenuated cell apoptosis by MAN, indicating that autophagy serves as cell death. Furthermore, autophagy-mediated cell death by MAN can be blocked by reactive oxygen species (ROS) scavenger NAC, indicating that ROS accumulation is the inducing factor of apoptosis and autophagy. In summary, we revealed the molecular mechanism of MAN against lung cancer through apoptosis and autophagy, suggesting that MAN might be a novel therapeutic agent for NSCLC treatment. [ABSTRACT FROM AUTHOR]

Hydrogen sulphide (H2S) is an important gasotransmitter with several physiological functions. However, the roles and the detailed mechanisms of H2S on skin wound healing are not known well. In the present study, 129S1/SvImJ mice were intraperitoneally injected with NaHS (50 μmol/kg/d) for 2 weeks. Then, a round wound of 6 mm diameter with depth into the dermis was made. The skin wound area, blood perfusion, superoxide production, malondialdehyde (MDA) levels, total antioxidant capacity (T‐AOC), expression of vascular endothelial growth factor (VEGF), dynamin‐related protein 1 (DRP1) and optic atrophy 1 (OPA1) were measured. After NaHS (50 μmol/L) pre‐administration for 4 hours, cell migration rate, DRP1, OPA1 and α–smooth muscle actin (α‐SMA) expression, superoxide production and mitochondrial membrane potential in primary skin fibroblasts were measured. Tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in human keratinocytes were also measured. The results showed that NaHS pretreatment significantly accelerated wound healing and improved blood flow in the wound after operation. NaHS increased VEGF expression in the wound and promoted tube formation in HUVECs. Meanwhile, NaHS attenuated reactive oxygen species (ROS) production, suppressed MDA level but restored T‐AOC in the wound. NaHS also promoted skin fibroblasts migration and α‐SMA expression after scratch. Moreover, NaHS alleviated ROS, increased mitochondrial membrane potential, decreased DRP1 but enhanced OPA1 expression in skin fibroblasts after scratch. NaHS also accelerated human keratinocytes migration after scratch. Taken together, exogenous H2S supplementary accelerated the skin wound healing, which might be related to oxidative stress inhibition and VEGF enhancement. [ABSTRACT FROM AUTHOR]

Dihydromyricetin (DMY) is one of the most important flavonoids in vine tea, which showed several pharmacological effects. However, information about the potential role of DMY on angiotensin II (Ang II) induced cardiac fibroblasts proliferation remains unknown. In the present study, cardiac fibroblasts isolated from neonatal Sprague-Dawley rats were pretreated with different concentrations of DMY (0–320 μM) for 4 h, or DMY (80 μM) for different time (0–24 h), followed by Ang II (100 nM) stimulation for 24 h, Then number of cardiac fibroblasts and content of hydroxyproline was measured. The level of cellular reactive oxygen species, malondialdehyde (MDA), activity of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were also evaluated. Expression of type I, type III collagen, α-smooth muscle actin (α-SMA), p22 phox (one vital subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase), SOD and thioredoxin (Trx) were detected with real time PCR or/and western blot. We found that pre-incubation with DMY (20 μM, 40 μM, 80 μM) for 4 h, 12 h or 24 h attenuated the proliferation of cardiac fibroblasts induced by Ang II. Expression of type I and type III collagen, as well as α-SMA were inhibited by DMY at both mRNA and protein level. DMY also significantly decreased cellular reactive oxygen species production and MDA level, while increased the SOD activity and T-AOC. DMY suppressed p22 phox , while enhanced antioxidant SOD and Trx expression in Ang II stimulated cardiac fibroblasts. Thus, dihydromyricetin attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress. [ABSTRACT FROM AUTHOR]