Methyloleanolate Induces Apoptotic And Autophagic Cell Death Via Reactive Oxygen Species Generation And c-Jun N-terminal Kinase Phosphorylation.

BACKGROUND: To develop a potent anticancer agent similar to oleanolate, the underlying mechanisms of its derivative, methyloleanolate, in the apoptosis and autophagy of A549 and H1299 cells were elucidated. PURPOSE: The aim of the present study was to investigate the effect of methyloleanolate in inducing apoptotic and autophagic cell death in cancer cells. MATERIALS AND METHODS: Flow cytometric analysis with Annexin V/PI staining, Western blot analysis, and immunofluorescence analysis were conducted in A549 and H1299 cells. RESULTS: Methyloleanolate increased the fraction of Annexin V/PI apoptotic cells and activated caspase-8, caspase-3, and death receptor 5 (DR5) more than oleanolate in A549 and H1299 cells pretreated with pancaspase inhibitor z-VAD-fmk and DR5 depletion. Also, methyloleanolate induced autophagic features of microtubule-associated protein light chain 3 3BII (LC3BII) conversion and puncta in A549 and H1299 cells, along with autophagosomes and vacuoles. Methyloleanolate blocked autophagy flux for impaired autophagy and chloroquine (CQ)-enhanced microtubule-associated protein LC3BII accumulation and cytotoxicity in A549 and H1299 cells, although 3-methyladenine (3-MA) did not. Interestingly, LC3BII accumulation was detected only in methyloleanolate-treated autophagy-related gene 5 (ATG5)+/+ mouse embryonic fibroblast (MEF) cells but not in ATG5 -/- MEF cells. Methyloleanolate reduced p-mTOR but activated p-c-Jun N-terminal kinases and reactive oxygen species production in A549 and H1299 cells. Conversely, n-acetyl-l-cysteine and SP600125 blocked apoptotic and autophagic cascades caused by methyloleanolate in A549 and H1299 cells. CONCLUSION: Overall, the findings suggest that methyloleanolate induces apoptotic and autophagic cell death in non-small cell lung cancers via reactive oxygen species generation and c-Jun N-terminal kinase phosphorylation.

Correction: CNOT2 promotes degradation of p62/SQSTM1 as a negative regulator in ATG5 dependent autophagy.

[This corrects the article DOI: 10.18632/oncotarget.17682.].

CNOT2 promotes degradation of p62/SQSTM1 as a negative regulator in ATG5 dependent autophagy.

Though CNOT2 is involved in regulation of adipogenic differentiation, apoptotic cell death and metastasis, the underlying autophagic mechanism of CNOT2 was unknown until now. Thus, in the present study, the critical role of CNOT2 in autophagy was elucidated in association with p62/SQSTM1 signaling. CNOT2 depletion induced p62/SQSTM1 accumulation and LC3B-II conversion, and also increased the number of puncta with impaired autophagic flux. In contrast, CNOT2 overexpression induced downregulation and ubiquitination of p62/SQSTM1 in HEK293 QBI. Furthermore, ubiquitination of p62/SQSTM1 was blocked by autophagy inhibition. Interestingly, CNOT2 was correlated with p62/SQSTM1 in HEK293 QBI cells and also was colocalized with p62/SQSTM1 in H1299 cells. Additionally, ATG5 was upregulated in CNOT2-depleted H1299 cells, while degradation of p62/SQSTM1 by CNOT2 was detected in ATG5+/+ MEF cells but not in ATG5-/- MEF cells. Of note, CNOT2 induced degradation of p62/SQSTM1 in HEK293 QBI cells co-transfected with Myc-ΔLIR/KIR or Myc-ΔUBA, but not with Myc-ΔPB1. Sub G1 population was increased in CNOT2-depleted H1299 cells by late autophagy inhibitors, ammonium chloride and chloroquine compared to 3-methyladenine. Overall, these findings provide novel insight into the critical role of CNOT2 as a negative regulator in ATG5 dependent autophagy.

Antiangiogenic Effect of Ethanol Extract of Vigna angularis via Inhibition of Phosphorylation of VEGFR2, Erk, and Akt.

Though dietary azuki bean (Vigna angularis) seed containing antioxidant proanthocyanidins was known to have multibiological activities including antioxidant, hypotensive, anti-inflammatory, and immunomodulatory activities, the antiangiogenic activity of ethanol extract of Vigna angularis (EVA) was never reported so far. In the present study, the antiangiogenic mechanism of EVA was examined in human umbilical vein endothelial cells (HUVECs). EVA showed weak cytotoxicity in HUVECs, while it significantly suppressed the VEGF induced proliferation of HUVECs. Consistently, wound healing assay revealed that EVA inhibited the VEGF induced migration of HUVECs. Also, EVA abrogated the VEGF induced tube formation of HUVECs in a concentration dependent fashion. Furthermore, Matrigel plug assay showed that EVA significantly reduced the hemoglobin level of Matrigel plug in mice compared to untreated control. Of note, EVA effectively attenuated the phosphorylation of VEGFR2, Erk, and Akt in VEGF-treated HUVECs. Overall, our findings suggest that EVA inhibits angiogenesis in VEGF-treated HUVECs via inhibition of phosphorylation of VEGFR2, ERK, and Akt.

A derivative of epigallocatechin-3-gallate induces apoptosis via SHP-1-mediated suppression of BCR-ABL and STAT3 signalling in chronic myelogenous leukaemia.

BACKGROUND AND PURPOSE: Epigallocatechin-3-gallate (EGCG) is a component of green tea known to have chemo-preventative effects on several cancers. However, EGCG has limited clinical application, which necessitates the development of a more effective EGCG prodrug as an anticancer agent. EXPERIMENTAL APPROACH: Derivatives of EGCG were evaluated for their stability and anti-tumour activity in human chronic myeloid leukaemia (CML) K562 and KBM5 cells. KEY RESULTS: EGCG-mono-palmitate (EGCG-MP) showed most prolonged stability compared with other EGCG derivatives. EGCG-MP exerted greater cytotoxicity and apoptosis in K562 and KBM5 cells than the other EGCG derivatives. EGCG-MP induced Src-homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) leading decreased oncogenic protein BCR-ABL and STAT3 phosphorylation in CML cells, compared with treatment with EGCG. Furthermore, EGCG-MP reduced phosphorylation of STAT3 and survival genes in K562 cells, compared with EGCG. Conversely, depletion of SHP-1 or application of the tyrosine phosphatase inhibitor pervanadate blocked the ability of EGCG-MP to suppress phosphorylation of BCR-ABL and STAT3, and the expression of survival genes downstream of STAT3. In addition, EGCG-MP treatment more effectively suppressed tumour growth in BALB/c athymic nude mice compared with untreated controls or EGCG treatment. Immunohistochemistry revealed increased caspase 3 and SHP-1 activity and decreased phosphorylation of BCR-ABL in the EGCG-MP-treated group relative to that in the EGCG-treated group. CONCLUSIONS AND IMPLICATIONS: EGCG-MP induced SHP-1-mediated inhibition of BCR-ABL and STAT3 signalling in vitro and in vivo more effectively than EGCG. This derivative may be a potent chemotherapeutic agent for CML treatment.

In vivo imaging of tumour bearing near-infrared fluorescence-emitting carbon nanodots derived from tire soot.

NIRF imaging of carbon nanodots derived from tire soot was clearly visualized in glioma in vitro and in vivo.

Bioimaging of targeting cancers using aptamer-conjugated carbon nanodots.

Using the excellent emission properties of carbon nanodots, we have developed an aptamer-conjugated imaging probe for targeting cancers.

Radioisotope imaging of microRNA-9-regulating neurogenesis using sodium iodide sympoter.

Since microRNAs (miRNA, miR) are known to be critical in various cellular processes and diseases, non-invasive molecular imaging system for miRNA is very important for imaging cellular therapy and disease diagnosis. In this study, we developed a radionuclide imaging system for miR-9 using sodium iodide symporter (NIS). During neuronal differentiation of P 19 cells induced by the treatment of retinoic acid (RA), in vitro and in vivo imaging demonstrated that the expression and activity of NIS from the miR-9 NIS reporter gene was clearly repressed by the increased expression and functional activity of miR-9 that bound with the target sequences in the NIS reporter gene and resulted in destabilized the transcriptional activity of NIS gene, compared with the undifferentiated P19 cells. The decreased activity of NIS from the differentiated P19 cells resulted in low uptake of radionuclide and decreased radioisotope signals. The NIS reporter gene-based miRNA imaging system showed a great specificity of imaging miRNA biogenesis during cellular developments. The miRNA NIS reporter gene will provide high sensitive imaging for visualizing miRNA-regulating cellular developments and diseases.

Carbon nanodot-based self-delivering microRNA sensor to visualize microRNA124a expression during neurogenesis.

MicroRNAs (miRNAs, miRs) are recognized as regulators of gene expression related to cellular development and diseases. In this study, we developed a carbon nanodot (C-dot)-based miR124a molecular beacon (miR124a CMB). The C-dots were purified from candle soot (cC-dots) by thermal oxidation. The double-stranded DNA oligonucleotide containing a miR124a binding site and black hole quencher 1 (miR124a sensing oligo) was further conjugated with the cC-dots to form the miR124a CMB. P19 cells were incubated with the miR124a CMB to sense miR124a expression during neurogenesis. The physical properties of the cC-dots showed multi-color light emission with various excitation wavelengths, a broad size distribution ranging from 2 to 4 nm, a graphitic carbon core (sp2), an abundance of carboxyl groups on the surface, no evidence of cellular toxicity and a high level of self-promoted uptake into cells. The miR124a CMB showed great fluorescence quenching in the absence of miR124a. The miR124a CMB internalized into P19 cells successfully visualized a gradual increase in miR124a expression during neuronal differentiation by providing signal-on imaging activity acquired by the following mechanism: the miR124a, which was highly expressed during neurogenesis, was bound to the miR124a binding site, resulting in the detachment of the quencher from the miR124a CMB and producing fluorescence recovery. The miR124a CMB demonstrated great specificity for sensing miR124a biogenesis with the advantages of self-passivated carboxyl groups, no toxicity, and self-illumination and highly self-promoted cellular uptake which will make the sensing of other various miRNAs related to diseases easy, convenient and accurate.

Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system.

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N(2+)-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.