ABSTRACT
MiR-873/CDK3 has been shown to play a critical role in ERα signaling and tamoxifen resistance. Thus, targeting this pathway may be a potential therapeutic approach for the treatment of ER positive breast cancer especially tamoxifen resistant subtype. Here we report that Norcantharidin (NCTD), currently used clinically as an ani-cancer drug in China, regulates miR-873/CDK3 axis in breast cancer cells. NCTD decreases the transcriptional activity of ERα but not ERß through the modulation of miR-873/CDK3 axis. We also found that NCTD inhibits cell proliferation and tumor growth and miR-873/CDK3 axis mediates cell proliferation suppression of NCTD. More important, we found that NCTD sensitizes resistant cells to tamoxifen. NCTD inhibits tamoxifen induced the transcriptional activity as well ERα downstream gene expressions in tamoxifen resistant breast cancer cells. In addition, we found that NCTD restores tamoxifen induced recruitments of ERα co-repressors N-CoR and SMRT. Knockdown of miR-873 and overexpression of CDK3 diminish the effect of NCTD on tamoxifen resistance. Our data shows that NCTD regulates ERα signaling and tamoxifen resistance by targeting miR-873/CDK3 axis in breast cancer cells. This study may provide an alternative therapy strategy for tamoxifen resistant breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cyclin-Dependent Kinase 3/metabolism , Drug Resistance, Neoplasm/drug effects , Estrogen Receptor alpha/metabolism , MicroRNAs/genetics , Tamoxifen/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , Cyclin-Dependent Kinase 3/genetics , Estrogen Receptor alpha/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Nude , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Parthenolide (PTL) selectively ablates leukemia stem cells (LSCs). A series of PTL derivatives with modifications on C-14 of PTL was synthesized, and most of the derivatives showed high activities against HL-60 and KG1a. The most potent compound 6j exhibited IC50 values of 0.4 µM and 1.1 µM against KG1a and HL-60, respectively, which were 8.7 and 3.8 folds more potent than those of PTL, respectively. Moreover, compound 6j showed relatively low toxicity to normal cells (IC50 = 12.3 µM) comparing with its high anti-AML activity. The selectivity indexes for AML cells KG1a and HL-60 were 30.8 and 11.2, respectively. Preliminary study revealed that compound 6j could induce apoptosis of KG1a cells.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Drug Design , Leukemia, Myeloid, Acute/pathology , Sesquiterpenes/chemical synthesis , Sesquiterpenes/pharmacology , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Proliferation/drug effects , Chemistry Techniques, Synthetic , HL-60 Cells , Humans , Sesquiterpenes/chemistry , Structure-Activity RelationshipABSTRACT
Nannocystin A is a 21-membered cyclodepsipeptide showing remarkable anticancer properties. Described is the total synthesis of nannocystin A, which features an asymmetric vinylogous Mukaiyama aldol reaction for efficient assembly of the penultimate open-chain precursor and a pivotal intramolecular Heck cross-coupling for the final macrocyclization.
Subject(s)
Macrocyclic Compounds/chemical synthesis , Cyclization , Macrocyclic Compounds/chemistry , Molecular ConformationABSTRACT
Hepatitis B x-interacting protein (HBXIP), a co-factor of survivin, was originally identified by its binding with the C-terminus of the hepatitis B virus x protein (HBx). We have recently shown that HBXIP promotes the growth of both normal liver cells and hepatoma cells in vitro, but the molecular mechanisms of this have not been documented. In this study, we investigated the potential effects of HBXIP on the proliferation of HepG2 cells and the intracellular signaling pathway mediating these changes. Over-expression of the HBXIP gene promoted the proliferation of HepG2 cells, as shown by the MTT assay. We also showed that HBXIP induced cellular accumulation in the S phase concomitantly with up-regulation of cyclinD(1) and down-regulation of p21 and p53 levels. Moreover, HBXIP over-expression cells showed activation of the PI3K/Akt pathway; this activation was accompanied by an increase in phosphorylation of glycogen synthase kinase 3ß. LY294002, a specific inhibitor of PI3K, blocked HBXIP-stimulated Akt phosphorylation and suppressed the cell cycle promotion induced by HBXIP in HepG2 cells. The increase in cyclinD(1) protein levels induced by HBXIP was inhibited when cells were incubated with LY294002. In conclusion, our data suggest that the proliferation of HepG2 cells promoted by HBXIP is associated with activation of the PI3K/Akt signaling pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/pharmacology , Cell Proliferation/drug effects , Hep G2 Cells/drug effects , Phosphatidylinositol 3-Kinase/drug effects , Proto-Oncogene Proteins c-akt/drug effects , Signal Transduction/drug effects , Adaptor Proteins, Signal Transducing/physiology , Blotting, Western , Chromones/pharmacology , Cyclin D1/antagonists & inhibitors , Cyclin D1/metabolism , Enzyme Activation/drug effects , Flow Cytometry , Hep G2 Cells/physiology , Humans , Morpholines/pharmacology , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinase/physiology , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/physiology , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Up-Regulation/drug effectsABSTRACT
Bacillus nematocida is a Gram-positive bacterium capable of killing nematodes. Our recent studies identified an extracellular serine protease Bace16 in B. nematocida as a candidate of pathogenic factor in the infection against nematodes, which displayed a high similarity with the serine protease family subtilisin BPN', and the MEROPS ID is S08.034. To further confirm the roles that bace16 played in the mechanism of nematocidal pathogenesis, recombinant mature Bace16 (rm-Bace16) was expressed in Escherichia coli strain BL21 using pET-30 vector system. Bioassay experiments demonstrated that the purified recombinant protease had the ability to degrade nematode cuticles and kill nematodes. In addition, a bace16 knockout mutant of B. nematocida constructed by homologous recombination showed considerably lower proteolytic activity and less than 50% nematocidal activity than the wild-type strain. These results confirmed that Bace16 could serve as an important virulence factor during the infectious process.
Subject(s)
Bacillus/pathogenicity , Nematoda/microbiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Animals , Bacillus/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Assay , Gene Deletion , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , VirulenceABSTRACT
Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host's cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4-6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.
Subject(s)
Gram-Positive Bacteria/enzymology , Gram-Positive Bacteria/pathogenicity , Peptide Hydrolases/metabolism , Rhabditida/microbiology , Amino Acid Sequence , Animals , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , Gram-Positive Bacteria/genetics , Molecular Sequence Data , Peptide Hydrolases/genetics , Peptide Hydrolases/isolation & purification , Sequence Analysis, DNAABSTRACT
Brevibacillus laterosporus G4, which was isolated from soil sample, kills free-living nematodes (Panagrellus redivius) and plant-parasite nematodes (Bursaphelenchus xylophilus) and degrades their cuticle in previous bioassay. Our works for B. laterosporus G4 had demonstrated that an extracellular alkaline protease BLG4 played a key role as a pathogenic factor in infection against nematode. In this study, the nematicidal activity of BLG4 was further verified by an in vitro assay with purified recombinant BLG4. The encoding gene of BLG4 was cloned and showed high degree of homology with the subtilisin subclass of serine protease gene and another reported cuticle-degrading protease gene from nematophagous bacterium Bacillus sp. B16. Deletion of BLG4 by homologous recombinant had a significant effect on the pathogenicity of B. laterosporus. In infection assays the BLG4-deficient strain (BLG4-6) lost about 50% of its nematocidal activity and in toxicity tests the mortality rate of nematodes decreased with approximately 56% in comparison to wild-type strain. This is the first report analyzing the function of a subtilisin enzyme involved in bacterium against nematode at the molecular level, and it is possible to use B. laterosporus as a model to study host-parasite interaction and to gain detailed knowledge of the infection process.