[The Effect of SIRT5 Deletion on Recovery of Hematopoietic Stem Cells after Injury in Mouse].

OBJECTIVE: To investigate the effect of deacylase Sirtuin 5 in the recovery of hematopoietic stem cells (HSCs) after treated by 5-FU in mouse. METHODS: Flow cytometry was used to analyze the effect of SIRT5 deletion on the proportion of hematopoietic stem/progenitor cells (HSPCs) in bone marrow (BM), the proportion of T cells, B cells and myeloid cells (TBM) in peripheral blood (PB) and spleen, and the development of T cells in thymus. Mouse were treated with 5-FU to study the effect of SIRT5 deletion on the cell cycle, apoptosis and the proportion of HSPCs in BM. The effect of SIRT5 deletion on the proliferation of HSCs was analyzed by flow sorting in vitro. RESULTS: SIRT5 deletion did not affect the development of T cells in thymus and the proportion of TBM cells in PB and spleen compared with wild type mice. SIRT5 deletion increased proportion of HSPCs in BM. After 5-FU treatment, the proportion of HSCs in SIRT5 deletion mice was significant decreased (P < 0.05), the HSPC in SIRT5 deletion mice was activated from G0 to G1 phase (P < 0.05), and the proportion of early apoptosis increased (P < 0.05). By monoclonal culture in vitro, the ability of HSCs to form clones in SIRT5 deletion mice was decreased significantly (P < 0.05). CONCLUSION: SIRT5 deletion lead to a decreased the ability of HSCs to clone in vitro. SIRT5 deletion is not conducive to the recovery of HSPCs injury in mice under hematopoietic stress.

mTORC2 Facilitates Liver Regeneration Through Sphingolipid-Induced PPAR-α-Fatty Acid Oxidation.

BACKGROUND & AIMS: During liver regeneration after partial hepatectomy, the function and metabolic pathways governing transient lipid droplet accumulation in hepatocytes remain obscure. Mammalian target of rapamycin 2 (mTORC2) facilitates de novo synthesis of hepatic lipids. Under normal conditions and in tumorigenesis, decreased levels of triglyceride (TG) and fatty acids (FAs) are observed in the mTORC2-deficient liver. However, during liver regeneration, their levels increase in the absence of mTORC2. METHODS: Rictor liver-specific knockout and control mice underwent partial hepatectomy, followed by measurement of TG and FA contents during liver regeneration. FA metabolism was evaluated by analyzing the expression of FA metabolism-related genes and proteins. Intraperitoneal injection of the peroxisome proliferator-activated receptor α (PPAR-α) agonist, p53 inhibitor, and protein kinase B (AKT) activator was performed to verify the regulatory pathways involved. Lipid mass spectrometry was performed to identify the potential PPAR-α activators. RESULTS: The expression of FA metabolism-related genes and proteins suggested that FAs are mainly transported into hepatocytes during liver regeneration. The PPAR-α pathway is down-regulated significantly in the mTORC2-deficient liver, resulting in the accumulation of TGs. The PPAR-α agonist WY-14643 rescued deficient liver regeneration and survival in mTORC2-deficient mice. Furthermore, lipidomic analysis suggested that mTORC2 deficiency substantially reduced glucosylceramide (GluCer) content. GluCer activated PPAR-α. GluCer treatment in vivo restored the regenerative ability and survival rates in the mTORC2-deficient group. CONCLUSIONS: Our data suggest that FAs are mainly transported into hepatocytes during liver regeneration, and their metabolism is facilitated by mTORC2 through the GluCer-PPAR-α pathway, thereby establishing a novel role for mTORC2 in lipid metabolism.

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis.

BACKGROUND: P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PURPOSE: To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. STUDY DESIGN AND METHODS: Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. RESULTS: The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. CONCLUSION: Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.

Phosphatase CDC25B Inhibitors Produced by Basic Alumina-Supported One-Pot Gram-Scale Synthesis of Fluorinated 2-Alkylthio-4-aminoquinazolines Using Microwave Irradiation.

An efficient, environmentally benign, and inexpensive procedure has been developed for the synthesis of fluorinated 2-alkylthio-4-aminoquinazolines by microwave irradiation using basic alumina as a solid-support agent as well as a solid base. Notably, this protocol features improved energy efficiency, broad isothiourea substrate scope, easily available starting materials, and high atom efficiency and applicability toward gram-scale synthesis. Additionally, the target compounds were evaluated for the cytotoxic effect against human colon adenocarcinoma (HCT116 and HT29), human gastric cancer (SGC-7901), human lung adenocarcinoma (A549), and human hepatocyte carcinoma (HepG2) cells, and it was found that these compounds have excellent antitumor activities. Among them, compound 3e was found to be one of the most potent derivatives with IC50 values lower than 9.44 µM against five human tumor cell lines, making it more active than cisplatin (DDP). Furthermore, for the first time, the fluorinated 2-alkylthio-substituted 4-aminoquinazolines were identified as phosphatase CDC25B inhibitors.

An Isoquinolin-1(2H)-Imine Derivative Induces Cell Death via Generation of Reactive Oxygen Species and Activation of JNK in Human A549 Cancer Cells.

Compound 11-benzoyl-10-chloro-7,9-difluoro-6-imino-2,3,4,6-tetrahydro-1H-pyrimido[1,2-b]isoquinoline-8-carbonitrile (HC6h) is a novel polyhalo 1,3-diazaheterocycle fused isoquinolin-1(2H)-imines derivative, which displays good anticancer activity and low toxicity in vivo. However, the underlying anticancer mechanisms have not previously been identified. The proliferation of A549 was assessed by MTT assay. The reactive oxygen species (ROS) level was assessed in A549 with a H2 DCFDA probe. Mitochondrial membrane potential was measured using the JC-1 staining. Apoptosis were measured by annexin-V/PI assay and autophagy by acridine orange staining and GFP-LC3 fluorescence assay. The expression of autophagic and apoptotic proteins was determined by Western blot. The compound HC6h increased accumulation of vesicles, acridine orange-stained cells and LC3-II in A549 cells. Inhibition of compound HC6h-induced autophagy by bafilomycin A1 increased apoptosis. Compound HC6h enhanced activation of caspase-3, caspase-9 and PARP cleavage in A549 cells. Compound HC6h leads to the rapid generation of intracellular ROS. Moreover, compound HC6h induced phosphorylation of JNK and was conferred by the increased ROS levels. Furthermore, down-regulation of JNK attenuated autophagic and apoptotic effect in response to HC6h. The induction of ROS upon HC6h treatment leads to the activation of JNK that mediates autophagy and apoptosis in human A549 cancer cells. J. Cell. Biochem. 118: 4394-4403, 2017. © 2017 Wiley Periodicals, Inc.