Jung Jin Hwang

Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells.

PURPOSE To characterize and investigate the mechanism of chloroquine (CQ) retinotoxicity in human retinal pigment epithelium-derived ARPE-19 cells. METHODS Cultured ARPE-19 cells were exposed to 10 to 250 μM CQ, and cell death was quantified using a lactate dehydrogenase release assay. Autophagy was studied in ARPE-19 cells transfected with GFP-LC3. Lysosomes in living cells were stained and observed by live-cell confocal microscopy. RESULTS After exposure to CQ, ARPE-19 cells developed cytosolic vacuoles within 1 hour and underwent cell lysis within 24 hours. The levels of LC3-II, beclin-1 and, p62, as well as the number GFP-LC3- and RPF-LC3-positive autophagic vacuoles (AVs), increased after CQ treatment, indicating that autophagy was activated. However, lysosomal staining revealed that almost all AVs were separate from lysosomes; thus, fusion between AVs and lysosomes was completely blocked. In addition, the levels of ubiquitinated proteins and GFP-mHttp aggregates in ARPE-19 cells were increased by CQ, providing further evidence that autophagic degradation was inhibited. CONCLUSIONS CQ induces vacuole formation and cell death in ARPE-19 cells. Initially, vacuoles developed from enlarged lysosomes, followed by the activation of upstream steps in the autophagy pathway and the formation of LC3-positive AVs. Because CQ blocked the fusion of AVs with lysosomes, autophagic protein degradation was inhibited, indicating that CQ-induced retinotoxicity may be caused by the accumulation of potentially toxic ubiquitinated proteins.

Zinc and 4-Hydroxy-2-Nonenal Mediate Lysosomal Membrane Permeabilization Induced by H2O2 in Cultured Hippocampal Neurons

Lysosomal membrane permeabilization (LMP) is implicated in cancer cell death. However, its role and mechanism of action in neuronal death remain to be established. In the present study, we investigate the function of cellular zinc in oxidative stress-induced LMP using hippocampal neurons. Live-cell confocal microscopy with FluoZin-3 fluorescence showed that H2O2 exposure induced vesicles containing labile zinc in hippocampal neurons. Double staining with LysoTracker or MitoTracker disclosed that the majority of the zinc-containing vesicles were lysosomes and not mitochondria. H2O2 additionally augmented the 4-hydroxy-2-nonenal (HNE) adduct level in lysosomes. Intracellular zinc chelation with TPEN [tetrakis(2-pyridylmethyl)ethylenediamine] completely blocked both HNE accumulation and neuronal death. Interestingly, within 1 h after the onset of H2O2 exposure, some of zinc-loaded vesicles lost their zinc signals. Consistent with the characteristics of LMP, a lysosomal enzyme, cathepsin D, was released into the cytosol, and cathepsin inhibitors partially rescued neuronal death. We further examined the possibility that HNE or zinc mediates H2O2-triggered LMP. Similar to H2O2, exposure to HNE or zinc triggered lysosomal zinc accumulation and LMP. Moreover, isolated lysosomes underwent LMP when exposed to HNE or zinc, but not H2O2, supporting the direct mediation of LMP by HNE and/or zinc. The appearance of zinc-containing vesicles and the increases in levels of cathepsin D and HNE, were also observed in hippocampal neurons of rats after kainate seizures. Thus, under oxidative stress, neuronal lysosomes accumulate zinc and HNE, and eventually undergo LMP, which may constitute a key mechanism of oxidative neuronal death.

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.

Multifunctional hollow gold nanoparticles designed for triple combination therapy and CT imaging.

Hollow gold nanoparticles (HGNP) are a novel class of hybrid metal nanoparticles whose unique optical and morphological properties have spawned new applications including more effective cancer therapy. The shell thickness of HGNPs can tune the surface plasmon resonance to the near infrared light, resulting in photothermal ablation of tumors with optimal light penetration in tissue. The hollow cavity within a HGNP is able to accommodate a high payload of chemotherapeutic agents. They have also been used for enhancing radiosensitization in tumors during radiotherapy due to the high X-ray absorption capability of gold particles. However, no report has yet been published that utilize HGNPs for the triple combination therapy and CT imaging. In this study, we synthesized HGNPs which exhibit better response to radiation for therapy and imaging and demonstrated the effects of combined chemotherapy, thermal and radiotherapy. This combination strategy presented delayed tumor growth by 4.3-fold and reduced tumors weight by 6.8-fold compared to control tumors. In addition, we demonstrated the feasibility of HGNP as a CT imaging agent. It is expected that translating these capabilities to human cancer patients could dramatically increase the antitumor effect and potentially overcome resistance to chemotherapeutic agents and radiation.

TrkB mediates BDNF-induced potentiation of neuronal necrosis in cortical culture.

In the present study, the signaling mechanisms underlying the effect of brain-derived neurotrophic factor (BDNF) on neuronal necrosis were investigated. Exposure of mature mouse cortical cultures (more than 10 days in vitro (DIV)) to 50-100 ng/ml BDNF for 48 h induced widespread neuronal necrosis that was antioxidant-sensitive. This neuronal necrosis was blocked by the selective NMDA antagonist MK-801, suggesting that prolonged BDNF exposure caused endogenous levels of NMDA receptor activation to become excitotoxic. We examined whether the p75(NTR) played a role in BDNF-induced neuronal death. However, p75(NTR) expression was low in cultured cortical cells, and neutralizing antibodies to p75(NTR) did not attenuate BDNF-triggered neuronal death. In contrast, trkB antisense oligonucleotides and inhibitors of Trk tyrosine kinase blocked BDNF-triggered neuronal death as well as BDNF potentiation of iron-induced oxidative neuronal necrosis, suggesting a critical role for TrkB in this phenomenon. Furthermore, BDNF did not potentiate neuronal necrosis in cortical cultures prepared from embryonic TrkB-null mice. These results suggest that TrkB plays an important role in BDNF-mediated neuronal necrosis.

Accumulation of labile zinc in neurons and astrocytes in the spinal cords of G93A SOD-1 transgenic mice

Zinc dyshomeostasis may trigger oxidative stress, which is likely the key mechanism of neuronal death in amyotrophic lateral sclerosis (ALS), including familial forms such as G93A SOD-1 ALS. Since zinc binding by G93A SOD-1 is weaker than by normal SOD-1, we assessed whether labile zinc levels are altered in the spinal cords of G93A SOD-1 transgenic (Tg) mice. Whereas no zinc-containing cells were found in wild-type (WT) mice, neurons and astrocytes with high levels of labile zinc appeared in G93A SOD-1 Tg mice, in correlation with motoneuron degeneration. The level of HNE, an endogenous neurotoxic molecule, was increased around zinc-accumulating cells and mSOD-1 positive cells, suggesting a link between HNE, SOD-1 mutation and zinc accumulation. Moreover, exposure of cultured spinal neurons and astrocytes from G93A SOD-1 Tg mice to HNE increased labile zinc levels, and exposure to zinc increased 4-hydroxynonenal (HNE) levels, to a greater degree than in WT neurons and astrocytes. Administration of the zinc chelator TPEN extended survival in G93A SOD-1 Tg mice. These results indicate that zinc dyshomeostasis occurs in the spinal cords of Tg mice, and that this dyshomeostasis may contribute to motoneuron degeneration.

Histone Deacetylase Inhibitor Potentiates Anticancer Effect of Docetaxel via Modulation of Bcl-2 Family Proteins and Tubulin in Hormone Refractory Prostate Cancer Cells

PURPOSE We evaluated the antitumor effects of docetaxel (Sigma®) and histone deacetylase inhibitors in hormone refractory prostate cancer cells, and analyzed the mechanism by which combination treatment induced cell death. MATERIALS AND METHODS We used LNCaP, DU145 and PC3 cells (ATCC®) to evaluate the in vitro apoptotic effects of histone deacetylase inhibitors and their combinations with docetaxel as well as the molecular mechanisms. The DU145 xenograft model was used to evaluate the in vivo efficacy of PXD101 combined with docetaxel. RESULTS Suberoylanilide hydroxamic acid or PXD101 inhibited the growth of hormone dependent LNCaP cells, and hormone independent DU145 and PC3 cells. It increased sub-G1 population and activated caspase-8, 9 and 3, indicating apoptosis induction. Pretreating DU145 cells with docetaxel followed by histone deacetylase inhibitors showed significant synergistic cytotoxicity compared with that of simultaneous co-treatment or reverse sequential treatment. Pretreatment with docetaxel followed by histone deacetylase inhibitors increased the apoptotic sub-G1 population, caspase activation and tubulin acetylation compared with that of docetaxel alone. Combination treatment decreased Mcl-1 and Bcl-xl, and increased t-Bid, Bik and Bim. Combined docetaxel and PXD101 reduced tumor size with efficacy equivalent to that of a double dose of docetaxel alone in the DU145 xenograft model. CONCLUSIONS These preclinical results indicate that the sequential combination of docetaxel and histone deacetylase inhibitors led to a synergistic increase in the death of hormone refractory prostate cancer cells via intrinsic and extrinsic apoptotic pathways by modulating Bcl-2 family proteins and tubulin in vitro and in vivo. Results suggest that this combination may be a new therapeutic modality in patients with hormone refractory prostate cancer.

Down-regulation of Mortalin Exacerbates Aβ-mediated Mitochondrial Fragmentation and Dysfunction

Background: Mitochondrial dysfunction is associated with neuronal disorders, and mitochondrial dynamics are altered in neurodegenerative diseases. Results: Inhibition of mortalin potentiates amyloid-β-mediated mitochondrial dysfunction and cytotoxicity. Conclusion: Inhibition of mortalin could lead to mitochondrial dysfunction through mitochondrial fragmentation. Significance: Activation of mortalin may antagonize the progression of Aβ-mediated neuronal injury in which mitochondrial dysfunction has a key role. Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aβ-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aβ-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aβ-mediated mitochondrial fragmentation and dysfunction in AD.

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

Alteration of the Cerebral Zinc Pool in a Mouse Model of Alzheimer Disease

Abstract Synaptic vesicle Zn2+ is regulated by zinc transporter 3 (ZnT3) and is involved in neurotransmission and synaptic plasticity. Here, we describe extensive alterations of ZnT3-regulated Zn2+ pools in the brains of human amyloid precursor protein-transgenic (Tg2576) mice. In contrast to wild-type littermates in which ZnT3 expression and synaptic Zn2+ increased with age, there were age-dependent reductions in ZnT3 expression and synaptic Zn2+ levels in the hippocampal mossy fiber area of Tg2576 mice. In these mice, a novel Zn2+ pool and ZnT3 expression were colocalized and appeared along dystrophic neurites surrounding compact amyloid plaques that were identified by in situ blue fluorescence, congophilic birefringence, and A&bgr;42 immunoreactivity. Zn2+-specific histofluorescence and ZnT3 immunofluorescence in dystrophic neurites were also colocalized with the &dgr;-subunit of adaptor protein complex 3, lysosome-associated membrane protein, cathepsin D, and neurofilament-containing hyperphosphorylated paired helical filaments. The synaptic vesicle marker protein synaptophysin and vesicle-associated membrane protein were not found in these neurites, suggesting a role of ZnT3 distinct from itsnormal role in synaptic Zn2+. ZnT3 immunoreactivity and Zn2+ histofluorescence were also evident in activated astrocytes. These datasuggest that extensive modifications of the cerebral Zn2+ pool, particularly synaptic Zn2+, may underlie neuronal dysfunction characteristic of Alzheimer disease.