Junseong Park

Mitochondrial Network Determines Intracellular ROS Dynamics and Sensitivity to Oxidative Stress through Switching Inter-Mitochondrial Messengers

Oxidative stresses caused by reactive oxygen species (ROS) can induce rapid depolarization of inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation. Damaged mitochondria produce more ROS, especially the superoxide anion (O2 −) and hydrogen peroxide (H2O2), which potentiate mitochondria-driven ROS propagation, so-called ROS-induced ROS release (RIRR), via activation of an inter-mitochondria signaling network. Therefore, loss of function in only a fraction of mitochondria might eventually affect cell viability through this positive feedback loop. Since ROS are very short-lived molecules in the biological milieu, mitochondrial network dynamics, such as density, number, and spatial distribution, can affect mitochondria-driven ROS propagation. To address this issue, we developed a mathematical model using an agent-based modeling approach, and tested the effect of mitochondrial network dynamics on RIRR for mitochondria under various conditions. Simulation results show that the intracellular ROS signaling pattern, such as ROS propagation speed and oxidative stress vulnerability, are critically affected by mitochondrial network dynamics. Mitochondrial network dynamics of mitochondrial distribution, density, activity, and size can mediate inter-mitochondrial signaling under certain conditions and determine the identity of the ROS signaling pattern. We further elucidated the potential mechanism of these actions, i.e., conversion of major messenger molecules involved in ROS signaling. If the average distance between neighboring mitochondria is large or mitochondrial distribution becomes randomized, messenger molecule of the ROS signaling network can be switched from O2 − to H2O2. In this case, mitochondria-driven ROS propagation is efficiently blocked by introduction of excess cytosolic glutathione peroxidase 1, while introduction of cytosolic superoxide dismutase has no effect. Together, these results suggest that mitochondrial network dynamics is a major determinant for cellular responses to RIRR through changing the key messenger molecules.

Hepatitis C virus infection enhances TNFα-induced cell death via suppression of NF-κB†

Hepatitis C virus (HCV) infection results in liver injury and long‐term complications, such as liver cirrhosis and hepatocellular carcinoma. Liver injury in HCV infection is believed to be caused by host immune responses, not by viral cytopathic effects. Tumor necrosis factor‐alpha (TNF‐α) plays a pivotal role in the inflammatory processes of hepatitis C. TNF‐α induces cell death that can be ameliorated by nuclear factor kappaB (NF‐κB) activation. We investigated the regulation of TNF‐α signal transduction in HCV‐infected cells and identified HCV proteins responsible for sensitization to TNF‐α‐induced cell death. We studied the effect of HCV infection on TNF‐α signal transduction using an in vitro HCV infection model (JFH‐1, genotype 2a) with Huh‐7 and Huh‐7.5 cells. We found that TNF‐α‐induced cell death significantly increased in HCV‐infected cells. HCV infection diminished TNF‐α‐induced phosphorylation of IκB kinase (IKK) and inhibitor of NF‐κB (IκB), which are upstream regulators of NF‐κB activation. HCV infection also inhibited nuclear translocation of NF‐κB and expression of NF‐κB‐dependent anti‐apoptotic proteins, such as B‐cell lymphoma—extra large (Bcl‐xL), X‐linked inhibitor of apoptosis protein (XIAP), and the long form of cellular‐FLICE inhibitory protein (c‐FLIP). Decreased levels of Bcl‐xL, XIAP, and c‐FLIP messenger RNA and protein were also observed in livers with chronic hepatitis C. Transfection with plasmids encoding each HCV protein revealed that core, nonstructural protein (NS)4B, and NS5B attenuated TNF‐α‐induced NF‐κB activation and enhanced TNF‐α‐induced cell death. Conclusion: HCV infection enhances TNF‐α‐induced cell death by suppressing NF‐κB activation through the action of core, NS4B, and NS5B. This mechanism may contribute to immune‐mediated liver injury in HCV infection. (HEPATOLOGY 2012;56:831–840)

Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin

Although curcumin suppresses the growth of a variety of cancer cells, its poor absorption and low systemic bioavailability have limited its translation into clinics as an anticancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated, more stable analog of curcumin, is significantly more potent than curcumin in inducing cell death and reducing the clonogenicity of malignant breast cancer cells. Furthermore, DMC reduces the tumor growth of xenografted MDA-MB 435S cells more strongly than curcumin. We found that DMC induces paraptosis accompanied by excessive dilation of mitochondria and the endoplasmic reticulum (ER); this is similar to curcumin, but a much lower concentration of DMC is required to induce this process. DMC inhibits the proteasomal activity more strongly than curcumin, possibly causing severe ER stress and contributing to the observed dilation. DMC treatment upregulates the protein levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and Noxa, and the small interfering RNA-mediated suppression of CHOP, but not Noxa, markedly attenuates DMC-induced ER dilation and cell death. Interestingly, DMC does not affect the viability, proteasomal activity or CHOP protein levels of human mammary epithelial cells, suggesting that DMC effectively induces paraptosis selectively in breast cancer cells, while sparing normal cells. Taken together, these results suggest that DMC triggers a stronger proteasome inhibition and higher induction of CHOP compared with curcumin, giving it more potent anticancer effects on malignant breast cancer cells.

Phenotypic Modulation of Primary Vascular Smooth Muscle Cells by Short-Term Culture on Micropatterned Substrate

Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing differentiation of VSMCs so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micropatterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micropatterned groove substrate to modulate the morphology and function of VSMCs. Later than 7th passage of VSMCs showed typical synthetic phenotype characterized by epithelial morphology, increased proliferation rates and corresponding gene expression profiles; while short-term culture of these cells on a micropatterned groove induced a change to an intermediate phenotype characterized by low proliferation rates, increased migration, a spindle-like morphology associated with cytoskeletal rearrangement and expression of muscle-specific genes. Microarray analysis showed preferential expression of contractile and smooth muscle cell-specific genes in cells cultured on the micropatterned groove. Culture on a patterned groove may provide a valuable model for the study the role of VSMCs in normal vascular physiology and a variety of proliferative vascular diseases.

Joint Time-Frequency Analysis of Radar Micro-Doppler Signatures from Aircraft Engine Models

Micro-Doppler signatures from four CAD aircraft engine models and one experimental engine model are analyzed using joint time-frequency analysis (JTFA). The signatures are obtained from shooting and bouncing rays (SBR) simulation for the CAD models and from measurement for the experimental model. The JTFA results give additional useful physical feature information regarding the respective engines, such as whether the number of blades is odd or even and the length of blades, in addition to the number of blades, which can be extracted by traditional spectrum analysis.

Treatment of Epidermal Growth Factor Receptor Inhibitor-Induced Acneiform Eruption with Topical Recombinant Human Epidermal Growth Factor

Background: Epidermal growth factor receptor (EGFR) inhibitors have been used as anticancer agents for the treatment of a variety of solid tumors. Related skin toxicities are the most common adverse effects and occur with all EGFR inhibitors. Several treatment approaches, such as antiseptic soaps, topical and oral antibiotics, and topical and oral corticosteroids, have been reported; however, the responses have been varied. Acneiform eruption induced by EGFR inhibitor treatment results from disturbed normal keratinocyte and hair follicle biology and may therefore benefit from local restoration of EGF pathway. Observations: We treated HaCaT cells with EGFR inhibitor and evaluated the expression of EGFR. After treatment of cells with EGFR inhibitor, EGFR expression was increased in a dose-dependent manner. We hypothesized that newly synthesized EGFR, not inhibited by EGFR inhibitors, may perform their biological action in keratinocytes in the presence of additional EGF. In this study, we therefore treated acneiform eruption patients with topical recombinant human EGF (rhEGF) with institutional review board approval. Here, we report three cases of such eruptions who responded to topical rhEGF. Conclusion: Topical rhEGF may be an effective treatment option for EGFR inhibitor-induced acneiform eruption.

TGF-β1 and hypoxia-dependent expression of MKP-1 leads tumor resistance to death receptor-mediated cell death.

Sporadic occurrence of transformed tumor cells is under the surveillance of the host immune system and such cells are effectively eliminated by immune-mediated cell death. During tumor progression, the antitumor effects of the tumor microenvironment are suppressed by diverse immunosuppressive mechanisms. In this research, we suggest novel immune evasion strategy of tumor cells through a transforming growth factor (TGF)-β1- and hypoxia-dependent mechanism. Experimental results showed that TGF-β1 and hypoxia induced mitogen-activated protein kinase phosphatase (MKP)-1 expression within 1 h, resulting in attenuation of c-Jun N-terminal kinase (JNK) phosphorylation and subsequent death receptor-mediated cell death. In addition, analysis of microarray data and immunostaining of MKP-1 in hepatocellular carcinoma (HCC) patient samples revealed that expression of MKP-1 is notably higher in tumors than in normal tissues, implying that MKP-1-dependent suppression of immune-mediated cell death takes place only in the tumor. To prove that MKP-1 can act as a mediator of immune escape by tumors, we determined whether chemo-resistance against several anticancer drugs could be overcome by knockdown of MKP-1. Cytotoxic assays showed that chemotherapy with siRNA targeting MKP-1 was significantly more effective than chemotherapy in the presence of MKP-1. Thus, we conclude that TGF-β1 and hypoxia ensure tumor cell survival and growth through expression of MKP-1.

Contribution of mitochondrial network dynamics to intracellular ROS signaling

Oxidative stresses can induce rapid depolarization of inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation. Damaged mitochondria produce more reactive oxygen species (ROS), particularly the superoxide anion (O2-) and hydrogen peroxide (H2O2), which potentiate mitochondria-driven ROS propagation, so-called ROS-induced ROS release (RIRR), via activation of an inter-mitochondrial signaling network. In this context, mitochondrial network dynamics, such as their density, number, and spatial distribution, can affect mitochondria-driven ROS propagation. To investigate this inter-mitochondrial communication, we developed a mathematical model using an agent-based modeling approach, and tested the effect of mitochondrial network dynamics on RIRR for mitochondria under various conditions. Simulation results show that mitochondrial network dynamics are critical determinants of inter-mitochondrial ROS signaling patterns and main messenger ROS molecules. We further elucidated the potential mechanism of these actions, which is conversion of major messenger molecules involved in ROS signaling. Collectively, we propose that mitochondrial network dynamics can determine cellular responses to oxidative stress by switching the molecular species involved in cellular signaling.

Constitutive Expression of MAP Kinase Phosphatase-1 Confers Multi-drug Resistance in Human Glioblastoma Cells

Purpose Current treatment of glioblastoma after surgery consists of a combination of fractionated radiotherapy and temozolomide. However, it is difficult to completely remove glioblastoma because it has uncertain boundaries with surrounding tissues. Moreover, combination therapy is not always successful because glioblastoma has diverse resistances. To overcome these limitations, we examined the combined effects of chemotherapy and knockdown of mitogen-activated protein kinase phosphatase-1 (MKP-1). Materials and Methods We used ten different anti-cancer drugs (cisplatin, cyclophosphoamide, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, mitomycin C, and vincristine) to treat glioblastoma multiforme (GBM) cells. Knockdown of MKP-1 was performed using siRNA and lipofectamine. The basal level of MKP-1 in GBM was analyzed based on cDNA microarray data obtained from the Gene Expression Omnibus (GEO) databases. Results Anti-cancer drug-induced cell death was significantly enhanced by knockdown of MKP-1, and this effect was most prominent in cells treated with irinotecan and etoposide. Treatment with these two drugs led to significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in a time-dependent manner, while pharmacological inhibition of JNK partially inhibited drug-induced cell death. Knockdown of MKP-1 also enhanced drug-induced phosphorylation of JNK. Conclusion Increased MKP-1 expression levels could be the cause of the high resistance to conventional chemotherapeutics in human GBM. Therefore, MKP-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy.

SoxF Transcription Factors Are Positive Feedback Regulators of VEGF Signaling

RATIONALE Vascular endothelial growth factor (VEGF) signaling is a key pathway for angiogenesis and requires highly coordinated regulation. Although the Notch pathway-mediated suppression of excessive VEGF activity via negative feedback is well known, the positive feedback control for augmenting VEGF signaling remains poorly understood. Transcription factor Sox17 is indispensable for angiogenesis, but its association with VEGF signaling is largely unknown. The contribution of other Sox members to angiogenesis also remains to be determined. OBJECTIVE To reveal the genetic interaction of Sox7, another Sox member, with Sox17 in developmental angiogenesis and their functional relationship with VEGF signaling. METHODS AND RESULTS Sox7 is expressed specifically in endothelial cells and its global and endothelial-specific deletion resulted in embryonic lethality with severely impaired angiogenesis in mice, substantially overlapping with Sox17 in both expression and function. Interestingly, compound heterozygosity for Sox7 and Sox17 phenocopied vascular defects of Sox7 or Sox17 homozygous knockout, indicating that the genetic cooperation of Sox7 and Sox17 is sensitive to their combined gene dosage. VEGF signaling upregulated both Sox7 and Sox17 expression in angiogenesis via mTOR pathway. Furthermore, Sox7 and Sox17 promoted VEGFR2 (VEGF receptor 2) expression in angiogenic vessels, suggesting a positive feedback loop between VEGF signaling and SoxF. CONCLUSIONS Our findings demonstrate that SoxF transcription factors are indispensable players in developmental angiogenesis by acting as positive feedback regulators of VEGF signaling.