DRG2 knockdown induces Golgi fragmentation via GSK3ß phosphorylation and microtubule stabilization.

The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3ß (GSK3ß)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3ß. Tau, a target of GSK3ß, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3ß phosphorylation, and microtubule stability.

DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells.

The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Microtubule polymerization inhibitor exhibited a reduced response in shDRG2 cells. Using immunoprecipitation, we show that DRG2 interacts with tau, which regulates microtubule polymerization. Collectively, these data demonstrate that DRG2 may aid in affecting microtubule dynamics in HeLa cells.

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 [(3)H] 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.

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

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.

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.

The isolation and in situ identification of MSCs residing in loose connective tissues using a niche-preserving organ culture system.

Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs.

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.

Fibrin matrix-supported three-dimensional organ culture of adipose tissue for selective outgrowth, expansion, and isolation of adipose-derived stem cells.

Conventional systems for isolating adipose-derived stem cells (ASC) require enzymatic digestion of adipose tissue (AT), followed by monolayer culture to the enrich the stem cell population. However, these systems are hindered by low cell yields and a lack of reproducibility. The present study was aimed at developing a unique strategy for isolating ASC based on fibrin matrix-supported three-dimensional (3-D) organ culture of native AT. Furthermore, we tried to optimize the fibrin composition by adjusting the fibrinogen and thrombin concentrations to allow rapid outgrowth and proliferation of ASC in the 3-D fibrin matrix. Human cutaneous AT fragments were encapsulated within the fibrin matrix to construct a 3-D environment and cultured under dynamic conditions. During in vitro culture the fibrin matrix provided physical support for the AT and also allowed selective outgrowth of ASC from embedded AT fragments. In situ expanded outgrown cells were recovered from the fibrin matrix by selective fibrinolysis and propagated under monolayer culture conditions. The cultured cells fulfilled the following criteria for ASC: adhesion to culture plastic, multipotent differentiation, correct immunophenotypic profile. Fibrin matrix-supported 3-D organ culture produced ASC that with high competency in terms of growth and differentiation capabilities, and resulted in a larger and more consistent cell yield than obtained with conventional culture systems. The fibrinogen and thrombin concentrations inversely affected spreading, migration, and ASC outgrowth from native AT. Our results indicate that this 3-D organ culture system for AT can be used as an efficient and reproducible method for ASC isolation.

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.

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 p21(Waf1/Cip1), p27(Kip1), and p15(INK4B) 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.

Anti-proliferative effect of Kv1.3 blockers in A549 human lung adenocarcinoma in vitro and in vivo.

Voltage-gated potassium (Kv) channels are widely expressed in the plasma membranes of numerous cells and contribute to a variety of cellular functions in both excitable neuronal cells and non-excitable epithelial cells. Recently, it has been demonstrated that Kv channels are associated with the proliferation of several types of cancer cells. In the present study, we investigated the effects of suppression of Kv1.3 expression on cell proliferation and cell cycle progression in human lung adenocarcinoma, A549 cells. Treatment with margatoxin (MgTX), a selective blocker of Kv1.3 or short hairpin RNA (shRNA) against Kv1.3, significantly blocked A549 cells' proliferation. In addition, selective inhibition of Kv1.3 significantly increased expression level of p21(Waf1/Cip1) and significantly decreased the expression level of Cdk4 and cyclin D3. We also applied the MgTX into a xenograft model using nude mice, and MgTX caused a reduction of tumor volume when it was injected into the tumor tissues. These results suggest that Kv1.3 may serve as a novel therapeutic target for lung adenocarcinoma therapy.

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.

Involvement of Kv4.1 K(+) channels in gastric cancer cell proliferation.

Voltage-gated potassium (Kv) channels are expressed not only in excitable cells but also in non-excitable cells such as epithelial cells. Recent studies have demonstrated that several subtypes of Kv channels are expressed in epithelial tumor cells, including human gastric cancer cells, and are associated with cell proliferation. In the present study, we examined the expression of Kv4.1 in human gastric cancer cell lines and the effects of suppressed expression of Kv4.1 on cell proliferation and cell cycle distribution. We found that Kv4.1 mRNA and protein are expressed in the human gastric cancer cell lines MKN-45 and SNU-638. Moreover, Kv4.1-targeted small interference RNA (siRNA) treatment inhibited gastric cancer cell proliferation. Flow cytometric analysis revealed that suppressed expression of Kv4.1 induced a G1-S transition block of cell cycle progression. These results reveal that Kv4.1 plays a role in the proliferation of the human gastric cancer cell lines MKN-45 and SNU-638 and can be considered as a therapeutic target for human gastric cancer.

Increased αB-crystallin in hypothalamic paraventricular nucleus of rats with myocardial infarction.

The hypothalamus plays an important role in maintaining a homeostasis of the body against stress response. In particular, the paraventricular nucleus of the hypothalamus is a critical region for disorders related to the autonomic nervous system, such as congestive heart failure and hypertension. αB-crystallin is a family of heat shock proteins that are widely expressed in the brain, including in glial cells, astrocytes, oligodendrocytes, and neurons. Many studies have demonstrated that expression level of αB-crystallin is up-regulated and involved in protecting cells from pathological conditions. In the present study, we examined the expression and potential role of αB-crystallin in the paraventricular nucleus (PVN) regions of rats with myocardial infarction (MI). Our results demonstrate that mRNA encoding αB-crystallin and protein for both native and phosphorylate forms (Ser-59) of αB-crystallin was significantly increased in the PVN during MI.

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 nu(s)(PO3(2-)) band intensity at ~970 cm(-1) and the phosphodiester symmetric stretching nu(s)(PO2(-)) band intensity at approximately 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.

In situ single cell monitoring by isocyanide-functionalized Ag and Au nanoprobe-based Raman spectroscopy.

The development of effective cellular imaging requires a specific labeling method for targeting, tracking, and monitoring cellular/molecular events in the living organism. For this purpose, we studied the cellular uptake of isocyanide functionalized silver and gold nanoparticles by surface enhanced Raman scattering (SERS). Inside a single mammalian cell, we could monitor the intracellular behavior of such nanoparticles by measuring the SERS spectra. The NC stretching band appeared clearly at approximately 2,100 cm-1 in the well-isolated spectral region from many organic constituents between 300 and 1,700 or 2,800 and 3,600 cm-1. The SERS marker band at approximately 2,100 cm-1 could be used to judge the location of the isocyanide-functionalized nanoparticles inside the cell without much spectral interference from other cellular constituents. Our results demonstrate that isocyanide-modified silver or gold nanoparticle-based SERS may have high potential for monitoring and imaging the biological processes at the single cell level.

Kv1.3 voltage-gated K(+) channel subunit as a potential diagnostic marker and therapeutic target for breast cancer.

Voltage-gated K(+) (Kv) channels are widely expressed in the plasma membranes of numerous cells such as epithelial cells. Recently, it has been demonstrated that Kv channels are associated with the proliferation of several types of cancer cells. Specifically, Kv1.3 seems to be involved in cancer cell proliferation and apoptosis. In the present study, we examined the expression of Kv1.3 in immortalized and tumorigenic human mammary epithelial cells. We also evaluated the expression level of Kv1.3 in each stage of breast cancer using mRNA isolated from breast cancer patients. In addition, treatment with tetraethylammonium, a Kv channel blocker, suppressed tumorigenic human mammary epithelial cell proliferation. Therefore, Kv1.3 may serve as a novel molecular target for breast cancer therapy while its stage-specific expression pattern may provide a potential diagnostic marker for breast cancer development.

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.

P2Y receptor regulation of sodium transport in human mammary epithelial cells.

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors. RT-PCR experiments also revealed expression of A(2b) adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (I(sc)) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na(+) channel (ENaC) alpha-, beta-, and gamma-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na(+) transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPgammaS. Basolateral stimulation with UTP produced a rapid but transient increase in I(sc) that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in I(sc) and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca(2+) mobilization evoked by P2Y receptor agonists stimulates Na(+) absorption by increasing the activity of Ca(2+)-activated K(+) channels located in the basolateral membrane.