Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in HepG2 hepatocellular carcinoma cells

AbstractApoptosis and autophagy are crucial mechanisms regulating cell death, and the relationship between apoptosis and autophagy in the liver has yet to be thoroughly explored. TIGAR (TP53-induced glycolysis and apoptosis regulator), which is a p53-inducible gene, functions in the suppression of ROS (reactive oxygen species) and protects U2OS cells from undergoing cell death. In this study, silencing TIGAR by RNAi (RNA interference) in HepG2 cells down-regulated both TIGAR mRNA (∼75%) and protein levels (∼80%) and led to the inhibition of cell growth (P<0.01) by apoptosis (P<0.001) and autophagy. We demonstrated that TIGAR can increase ROS levels in HepG2 cells. The down-regulation of TIGAR led to the induction of LC-3 II (specific autophagic marker), the formation of the autophagosome, and increased Beclin-1 expression. 3-MA (3-Methyladenine), an inhibitor of autophagic sequestration blocker, inhibited TIGAR siRNA-enhanced autophagy, as indicated by the decrease in LC-3 II levels. Consequently, these data provide the first evidence that targeted silencing of TIGAR induces apoptotic and autophagic cell death in HepG2 cells, and our data raise hope for the future successful application of TIGAR siRNA in patients with hepatocellular carcinoma (HCC)

Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II

CCCTC binding factor (CTCF) mutants that cannot bind components of the polycomb repressive complex-2 (PRC2) do not form the chromatin loops that regulate monoallelic gene expression

MiR-145, a new regulator of the DNA Fragmentation Factor-45 (DFF45)-mediated apoptotic network

<p>Abstract</p> <p>Background</p> <p>MicroRNA-145 (miR-145) is considered to play key roles in many cellular processes, such as proliferation, differentiation and apoptosis, by inhibiting target gene expression. DNA Fragmentation Factor-45 (DFF45) has been found to be the substrate of Caspase-3, and the cleavage of DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. There are currently no in-depth studies on the relationship between miR-145 and the DFF45 gene.</p> <p>Results</p> <p>In this study, we identified DFF45 as a novel target of miR-145. We demonstrated that miR-145 targets a putative binding site in the coding sequence (CDS) of DFF45, and its abundance is inversely associated with DFF45 expression in colon cancer cells. Using a luciferase reporter system, we found that miR-145 suppresses the expression of the luciferase reporter gene fused to the putative binding site of DFF45. The level of DFF45 protein, but not DFF45 mRNA, was decreased by miR-145, suggesting a mechanism of translational regulation. Furthermore, we demonstrate that this specific silencing of DFF45 by miR-145 accounts, at least in part, for the staurosporine-induced tumor cell apoptosis <it>in vitro</it>.</p> <p>Conclusions</p> <p>Our study reveals a previously unrecognized function of miR-145 in DFF45 processing, which may underlie crucial aspects of cancer biology.</p

Therapeutic efficacy by targeting correction of Notch1-induced aberrants in uveal tumors.

There is a need for more effective treatments for uveal melanoma. The recombinant oncolytic adenovirus H101 replicates specifically in p53-depleted tumor cells, and has been approved for use by the Chinese State Food and Drug Administration. However, this treatment is associated with subsequent remission. Transfection of uveal melanoma cells with a small interfering RNA against Notch1 (siNotch1) effectively suppressed Notch1 expression, resulting in significant cell growth inhibition when combined with H101 treatment. Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) greatly enhanced apoptosis and cell cycle arrest in vitro as compared to treatment with H101 or siNotch1 alone. For in vivo treatments, the combined treatment of siNotch1 and H101 showed remarkable tumor growth inhibition and prolonged mouse survival in the OCM1 xenograft model. We predict that Notch pathway deregulation could be a feature of uveal melanoma, and could be a therapeutic target, especially if p53 is concurrently targeted

Combined Treatment with an Oncolytic Adenovirus and Antitumor Activity of Vincristine against Retinoblastoma Cells

Abstract: Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB44 cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G2/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in theInt. J. Mol. Sci. 2012, 13 1073

Cell cycle distribution and apoptotic activity of combined H101-Notch1-siRNA on UM cells.

<p>(A) and (B) Cell cycle distribution of OCM1 and VUP cells following treatment with siNotch1 and/or H101. OCM1 and VUP cells were harvested 72 hours after co-treatment with siNotch1 (50nmol/L) and H101 (MOI = 100), and propidium iodide staining and FACS analysis were used to analyze the cell cycle distribution. S-phase arrest was detected in the H101 and H101-Notch1-siRNA groups. (C) and (D) Apoptotic activity of OCM1 and VUP cells. Cells were measured by flow cytometry analysis 72 hours after co-treatment with siNotch1 (50nmol/L) and/or H101 (MOI = 100). Upper left: cells affected by necrosis only; upper right: cells affected with both apoptosis and necrosis; lower left: normal cells; lower right: cells affected by apoptosis only. Data are expressed as mean ± SD. of three independent experiments. (*: p<0.05, **: p<0.01, compared with untreated tumor cells). (E) and (F) Relative ratio percentage of apoptosis (cells in lower right group) and necrosis (cells in upper left group) in OCM1 and VUP cells. The percentages of apoptosis and necrosis cells were analyzed according to (C) and (D).</p