Soo Hwa Jang

In vitro toxicity of serum protein-adsorbed citrate-reduced gold nanoparticles in human lung adenocarcinoma cells

We examined the cytotoxicity effect of the serum protein coated gold nanoparticles (AuNPs) in the A549 cells. Negatively charged AuNPs were prepared by chemical reduction using citrate. The dimension and surface charge of AuNPs were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements. The AuNPs modified by the citrate anion were presumed to adsorb the serum proteins as indicated from the visible absorption spectroscopy, DLS, and quartz crystal microbalance (QCM) data. The QCM results indicated that among the constituents, fetal bovine serum (FBS) should be the major adsorbate species on the AuNPs incubated in the RPMI medium. The internalization of AuNPs into the A549 cells was also monitored using TEM and dark-field microscopy (DFM). Both methylthiazol tetrazolium (MTT) and lactate dehydrogenase (LDH) assays revealed that AuNPs were toxic as determined by their half-maximal inhibitory concentration. A flow cytometric and real-time PCR analysis of apoptotic genes along with the ATP depletion measurements suggested that AuNPs induce cell damages through extrinsic and intrinsic apoptotic pathways.

Capsaicin suppresses the migration of cholangiocarcinoma cells by down-regulating matrix metalloproteinase-9 expression via the AMPK-NF-κB signaling pathway.

Cholangiocarcinoma is one of the most difficult malignancies to cure. An important prognostic factor is metastasis, which precludes curative surgical resection. Recent evidence shows that capsaicin has an inhibitory effect on cancer cell migration and invasion. Here, we investigated the molecular mechanism of the capsaicin-induced anti-migration and anti-invasion effects on HuCCT1 cholangiocarcinoma cells. Migration and invasion were significantly reduced in response to capsaicin. Capsaicin also inhibited the expression of matrix metalloproteinase-9 (MMP-9). In capsaicin-treated cells, levels of phosphorylated AMPK increased, and this effect was abolished by treatment with the AMPK inhibitor, Compound C. Capsaicin enhanced the expression of SIRT1, which can activate the transcription factor NF-κB by deacetylation. This suggests that NF-κB is activated by capsaicin via the SIRT1 pathway. In addition, capsaicin-activated AMPK induced the phosphorylation of IκBα and nuclear localization of NF-κB p65. Chromatin immunoprecipitation assays demonstrated that capsaicin reduced MMP-9 transcription by inhibiting NF-κB p65 translocation and deacetylation via SIRT1. These findings provide evidence that capsaicin suppresses the migration and invasion of cholangiocarcinoma cells by inhibiting NF-κB p65 via the AMPK–SIRT1 and the AMPK–IκBα signaling pathways, leading to subsequent suppression of MMP-9 expression.

Nuclear localization and functional characteristics of voltage-gated potassium channel Kv1.3.

Background: Voltage-gated K+ channels are generally known to be located in the plasma membrane. Results: Kv1.3 in the nucleus regulates nuclear membrane potential and activates transcription factors. Conclusion: Kv1.3 is a functional channel at the nucleus. Significance: This study provides novel insight about functional nuclear Kv1.3 and expands our knowledge about the function of ion channels within the nucleus. It is widely known that ion channels are expressed in the plasma membrane. However, a few studies have suggested that several ion channels including voltage-gated K+ (Kv) channels also exist in intracellular organelles where they are involved in the biochemical events associated with cell signaling. In the present study, Western blot analysis using fractionated protein clearly indicates that Kv1.3 channels are expressed in the nuclei of MCF7, A549, and SNU-484 cancer cells and human brain tissues. In addition, Kv1.3 is located in the plasma membrane and the nucleus of Jurkat T cells. Nuclear membrane hyperpolarization after treatment with margatoxin (MgTX), a specific blocker of Kv1.3 channels, provides evidence for functional channels at the nuclear membrane of A549 cells. MgTX-induced hyperpolarization is abolished in the nuclei of Kv1.3 silenced cells, and the effects of MgTX are dependent on the magnitude of the K+ gradient across the nuclear membrane. Selective Kv1.3 blockers induce the phosphorylation of cAMP response element-binding protein (CREB) and c-Fos activation. Moreover, Kv1.3 is shown to form a complex with the upstream binding factor 1 in the nucleus. Chromatin immunoprecipitation assay reveals that Sp1 transcription factor is directly bound to the promoter region of the Kv1.3 gene, and the Sp1 regulates Kv1.3 expression in the nucleus of A549 cells. These results demonstrate that Kv1.3 channels are primarily localized in the nucleus of several types of cancer cells and human brain tissues where they are capable of regulating nuclear membrane potential and activation of transcription factors, such as phosphorylated CREB and c-Fos.

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.

Dendrotoxin-κ suppresses tumor growth induced by human lung adenocarcinoma A549 cells in nude mice

Voltage-gated K+ (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ), on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-κ, reduced tumor formation in nude mice. Furthermore, treatment with DTX-κ significantly increased protein expression of p21Waf1/Cip1, p27Kip1, and p15INK4B and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-κ has anti-tumor effects in A549 cells through the pathway governing G1-S transition.

Infrared spectroscopy characterization of normal and lung cancer cells originated from epithelium.

The vibrational spectral differences of normal and lung cancer cells were studied for the development of effective cancer cell screening by means of attenuated total reflection infrared spectroscopy. The phosphate monoester symmetric stretching νs(PO32-) band intensity at ~970 cm-1 and the phosphodiester symmetric stretching νs(PO2-) band intensity at ~1,085 cm-1 in nucleic acids and phospholipids appeared to be significantly strengthened in lung cancer cells with respect to the other vibrational bands compared to normal cells. This finding suggests that more extensive phosphorylation occur in cancer cells. These results demonstrate that lung cancer cells may be prescreened using infrared spectroscopy tools.

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Adenosine reduces GABAergic IPSC frequency via presynaptic A1 receptors in hypothalamic paraventricular neurons projecting to rostral ventrolateral medulla

Adenosine is an inhibitory modulator of neuronal transmission, including GABAergic transmission in the hypothalamus. It is known that the local GABAergic inputs tonically inhibit the hypothalamic paraventricular neurons projecting to the rostral ventrolateral medulla (RVLM; PVN-RVLM neurons) which regulate sympathetic outflow. In this study, we examined the effects of adenosine on GABAergic synaptic transmission in the PVN-RVLM neurons using whole cell patch-clamp combined with the retrograde labeling technique. Adenosine (100 μM) reversibly decreased the frequency of miniature IPSCs (from 3.41 ± 0.75 to 2.19 ± 0.49 Hz) in a concentration-dependent manner (IC₅₀ = 1.0 μM) without affecting the amplitude and the decay time constant of miniature IPSCs. Adenosine increased the paired-pulse ratio of evoked IPSCs from 1.19 ± 0.05 to 2.28 ± 0.09 (P<0.001). The effects of adenosine was mimicked by a selective A₁ receptor agonist (CHA, 10 μM), and blocked by a selective A₁ receptor antagonist (DPCPX, 2 μM), but not by a selective A₂ receptor antagonist (DMPX, 10 μM). In conclusion, the results showed that adenosine inhibits synaptic GABA release via presynaptic A₁ receptors in the PVN-RVLM neurons, indicating a potential of adenosine A₁ receptors in regulating sympathetic tone in normal and disease states.

DRG2 Regulates G2/M Progression via the Cyclin B1-Cdk1 Complex.

Developmentally regulated GTP-binding protein 2 (DRG2) plays an important role in cell growth. Here we explored the linkage between DRG2 and G2/M phase checkpoint function in cell cycle progression. We observed that knockdown of DRG2 in HeLa cells affected growth in a wound-healing assay, and tumorigenicity in nude mice xenografts. Flow cytometry assays and [3H] incorporation assays indicated that G2/M phase arrest was responsible for the decreased proliferation of these cells. Knockdown of DRG2 elicited down-regulation of the major mitotic promoting factor, the cyclin B1/Cdk1 complex, but up-regulation of the cell cycle arresting proteins, Wee1, Myt1, and p21. These findings identify a novel role of DRG2 in G2/M progression.

Expression of Keratin 10 in Rat Organ Surface Primo-vascular Tissues

The primo-vascular system is described as the anatomical structure corresponding to acupuncture meridians and has been identified in several tissues in the body, but its detailed anatomy and physiology are not well understood. Recently, the presence of keratin 10 (Krt10) in primo-vascular tissue was reported, but this finding has not yet been confirmed. In this study, we compared Krt10 expression in primo-vascular tissues located on the surface of rat abdominal organs with Krt10 expression on blood and lymphatic vessels. Krt10 protein (approximately 56.5 kDa) was evaluated by western blot analysis and immunohistochemistry. Krt10 (IR) in the primo-node was visualized as patchy spots around each cell or as a follicle-like structure containing a group of cells. Krt10 IR was also identified in vascular and lymphatic tissues, but its distribution was diffuse over the extracellular matrix of the vessels. Thus Krt10 protein was expressed in all three tissues tested, but the expression pattern of Krt10 in primo-vascular tissue differed from those of blood and lymphatic vascular tissues, suggesting that structural and the regulatory roles of Krt10 in primo-vascular system are different from those in blood and lymphatic vessels.