naphthoquinones and 3-methyladenine

Juglone (JG), a naturally-occurring naphthoquinone of Manchurian walnut (Juglans mandshurica) was shown to inhibit proliferation in various tumor types. However, the molecular mechanisms of JG on the induction of apoptosis and autophagy in HepG2 cells have not been examined. Herein, we investigated that JG could inhibit cell proliferation by induction of G2/M phase arrest. Also, occurrence of apoptosis was closely related with loss of mitochondrial membrane potential, the changes of apoptosis-related proteins after treatment with JG. In addition, we found that JG caused autophagy, as evidenced by increased expressions of LC3-II and Beclin-1. Interestingly, inhibition of JG-induced autophagy by 3-methyladenine (3-MA) and wortmannin (WT) significantly decreased apoptosis, whereas the apoptosis inhibitor z-VAD-fmk slightly enhanced autophagy. Furthermore, the induction of autophagy and apoptosis was associated with activation of MAPK family members (p38 and JNK) and production of reactive oxygen species (ROS). Both JNK inhibitor (SP600125) and ROS scavenger (N-acetylcysteine, NAC) could attenuate JG-induced autophagy and apoptosis. However, the p38-specific inhibitor SB203580 enhanced autophagic and apoptotic death. Moreover, the ROS scavenger NAC prevented phosphorylation of both p38 and JNK. Collectively, our data revealed that JG induced G2/M phase arrest, apoptosis, and autophagy through the ROS-dependent signaling pathway.

Topics: Acetylcysteine; Adenine; Amino Acid Chloromethyl Ketones; Androstadienes; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Enzyme Activation; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Wortmannin

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.

Topics: Adenine; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Deoxycytidine; DNA Fragmentation; Drug Resistance, Neoplasm; Everolimus; Gemcitabine; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Microtubule-Associated Proteins; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Securin; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington's disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, β-lapachone (β-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with β-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). β-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, β-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. β-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by β-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of β-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, β-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that β-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, β-lap provides a novel therapeutic opportunity for the treatment of HD.

Topics: Adenine; Animals; Animals, Genetically Modified; Apoptosis; Autophagy; Blotting, Western; Caenorhabditis elegans; Cell Movement; Cell Proliferation; Green Fluorescent Proteins; Humans; Huntingtin Protein; Immunoenzyme Techniques; Immunoprecipitation; Microscopy, Fluorescence; Naphthoquinones; Nerve Tissue Proteins; Neuroblastoma; Peptides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sirtuin 1

Autophagy is mainly responsible for the degradation of long-lived proteins and subcellular organelles. Autophagy is responsible for the non-apoptotic cell death, and plays a crucial role in regulating cellular functions. β-Lapachone is a quinone-containing compound originally obtained from the lapacho tree in South America. Here, we show that β-lapachone induces death in U87 MG cells, which is not inhibited by blockers of pan-caspase or necrosis. β-Lapachone-induced cell death gradually increased in a time-dependent manner in U87 MG cells, which were partly prevented by pretreatment of a specific inhibitor of NQO1 (dicoumarol). These results suggested that β-lapachone-induced cell death was mediated by NQO1-independent as well as NQO1-dependent cell death pathways. During progression of β-lapachone-induced cell death, translocation and processing of LC3 as well as an increase in acidic vesicular organelles, as assessed by acridine orange staining, were observed. Furthermore, β-lapachone-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by autophagy inhibitors (3-methyl adenine or bafilomycin A1). Reactive oxygen species (ROS) were involved in β-lapachone-induced autophagic cell death of U87 MG glioma cells, because β-lapachone induced ROS production and antioxidant N-acetylcysteine (NAC) decreased autophagic cell death. Our results collectively demonstrate that ROS mediate β-lapachone-induced autophagic cell death in U87 MG glioma cells.

Topics: Adenine; Autophagy; Blotting, Western; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Flow Cytometry; Glioma; Humans; Macrolides; Microscopy, Fluorescence; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Reactive Oxygen Species; RNA, Small Interfering