Focused Ultrasound as a Novel Non-Invasive Method for the Delivery of Gold Nanoparticles to Retinal Ganglion Cells.

Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.

Cervical Myelopathy Due to Epidural Hematoma at the Cervicomedullary Junction Associated With Ventriculoperitoneal Shunt Overdrainage: A Case Report.

We present a case of cervical myelopathy caused by epidural hematoma formation due to chronic cerebrospinal fluid overdrainage. A 55-year-old man who underwent ventriculoperitoneal (V-P) shunt surgery for normal pressure hydrocephalus presented with progressive weakness of both the upper and lower extremities. Magnetic resonance imaging (MRI) revealed compressive myelopathy at the cervicomedullary junction at the C1-C2 level caused by epidural hematoma formation due to intracranial hypotension (IH) caused by a complication of V-P shunt. He underwent decompressive laminectomy and hematoma removal at C1-C2 and replacement of the V-P shunt valve. Follow-up cervical spine MRI showed an improved state of severe central spinal stenosis at the C1-C2 level and an improved state of compression-related cord signal intensity change in the spinal cord. After surgical intervention and intensive rehabilitation, the patient showed clinical improvement. If cervical myelopathy is suspected in patients with a shunt, cord compression due to venous engorgement or hematoma caused by over-shunting and IH should be considered.

Long-term results on the suppression of secondary brain injury by early administered low-dose baclofen in a traumatic brain injury mouse model.

Secondary injury from traumatic brain injury (TBI) perpetuates cerebral damages through varied ways. Attenuating neuroinflammation, which is a key feature of TBI, is important for long-term prognosis of its patients. Baclofen, a muscle relaxant, has shown promise in reducing excessive inflammation in other neurologic disorders. However, its effectiveness in TBI remains ambiguous. Thus, our study aimed to investigate whether early administration of baclofen could elicit potential therapeutic effects by diminishing exaggerated neuroinflammation in TBI mice. In this study, 80 C57BL/6 mice were used, of which 69 mice received controlled cortical impact. The mice were divided into six groups (11-16 mice each). Baclofen, administered at dose of 0.05, 0.2 and 1 mg/kg, was injected intraperitoneally a day after TBI for 3 consecutive weeks. 3 weeks after completing the treatments, the mice were assessed histologically. The results showed that mice treated with baclofen exhibited a significantly lower volume of lesion tissue than TBI mice with normal saline. Baclofen also reduced activated glial cells with neurotoxic immune molecules and inhibited apoptotic cells. Significant recovery was observed and sustained for 6 weeks at the 0.2 mg/kg dose in the modified neurological severity score. Furthermore, memory impairment was recovered with low-doses of baclofen in the Y-maze. Our findings demonstrate that early administration of low dose baclofen can regulate neuroinflammation, prevent cell death, and improve TBI motor and cognitive abnormalities.

Combined Effects of Focused Ultrasound and Photodynamic Treatment for Malignant Brain Tumors Using C6 Glioma Rat Model.

PURPOSE: Glioblastoma (GBM) is an intractable disease for which various treatments have been attempted, but with little effect. This study aimed to measure the effect of photodynamic therapy (PDT) and sonodynamic therapy (SDT), which are currently being used to treat brain tumors, as well as sono-photodynamic therapy (SPDT), which is the combination of these two. MATERIALS AND METHODS: Four groups of Sprague-Dawley rats were injected with C6 glioma cells in a cortical region and treated with PDT, SDT, and SPDT. Gd-MRI was monitored weekly and 18F-FDG-PET the day before and 1 week after the treatment. The acoustic power used during sonication was 5.5 W/cm² using a 0.5-MHz single-element transducer. The 633-nm laser was illuminated at 100 J/cm². Oxidative stress and apoptosis markers were evaluated 3 days after treatment using immunohistochemistry (IHC): 4-HNE, 8-OhdG, and Caspase-3. RESULTS: A decrease in tumor volume was observed in MRI imaging 12 days after the treatment in the PDT group (p<0.05), but the SDT group showed a slight increase compared to the 5-Ala group. The high expression rates of reactive oxygen species-related factors, such as 8-OhdG (p<0.001) and Caspase-3 (p<0.001), were observed in the SPDT group compared to other groups in IHC. CONCLUSION: Our findings show that light with sensitizers can inhibit GBM growth, but not ultrasound. Although SPDT did not show the combined effect in MRI, high oxidative stress was observed in IHC. Further studies are needed to investigate the safety parameters to apply ultrasound in GBM.

Enhanced delivery of a low dose of aducanumab via FUS in 5×FAD mice, an AD model.

BACKGROUND: Aducanumab (Adu), which is a human IgG1 monoclonal antibody that targets oligomer and fibril forms of beta-amyloid, has been reported to reduce amyloid pathology and improve impaired cognition after administration of a high dose (10 mg/kg) of the drug in Alzheimer's disease (AD) clinical trials. The purpose of this study was to investigate the effects of a lower dose of Adu (3 mg/kg) with enhanced delivery via focused ultrasound (FUS) in an AD mouse model. METHODS: The FUS with microbubbles opened the blood-brain barrier (BBB) of the hippocampus for the delivery of Adu. The combined therapy of FUS and Adu was performed three times in total and each treatment was performed biweekly. Y-maze test, Brdu labeling, and immunohistochemical experimental methods were employed in this study. In addition, RNA sequencing and ingenuity pathway analysis were employed to investigate gene expression profiles in the hippocampi of experimental animals. RESULTS: The FUS-mediated BBB opening markedly increased the delivery of Adu into the brain by approximately 8.1 times in the brains. The combined treatment induced significantly less cognitive decline and decreased the level of amyloid plaques in the hippocampi of the 5×FAD mice compared with Adu or FUS alone. Combined treatment with FUS and Adu activated phagocytic microglia and increased the number of astrocytes associated with amyloid plaques in the hippocampi of 5×FAD mice. Furthermore, RNA sequencing identified that 4 enriched canonical pathways including phagosome formation, neuroinflammation signaling, CREB signaling and reelin signaling were altered in the hippocami of 5×FAD mice receiving the combined treatment. CONCLUSION: In conclusion, the enhanced delivery of a low dose of Adu (3 mg/kg) via FUS decreases amyloid deposits and attenuates cognitive function deficits. FUS-mediated BBB opening increases adult hippocampal neurogenesis as well as drug delivery. We present an AD treatment strategy through the synergistic effect of the combined therapy of FUS and Adu.

Gold-like activity copper-like selectivity of heteroatomic transition metal carbides for electrocatalytic carbon dioxide reduction reaction.

An overarching challenge of the electrochemical carbon dioxide reduction reaction (eCO2RR) is finding an earth-abundant, highly active catalyst that selectively produces hydrocarbons at relatively low overpotentials. Here, we report the eCO2RR performance of two-dimensional transition metal carbide class of materials. Our results indicate a maximum methane (CH4) current density of -421.63 mA/cm2 and a CH4 faradic efficiency of 82.7% ± 2% for di-tungsten carbide (W2C) nanoflakes in a hybrid electrolyte of 3 M potassium hydroxide and 2 M choline-chloride. Powered by a triple junction photovoltaic cell, we demonstrate a flow electrolyzer that uses humidified CO2 to produce CH4 in a 700-h process under one sun illumination with a CO2RR energy efficiency of about 62.3% and a solar-to-fuel efficiency of 20.7%. Density functional theory calculations reveal that dissociation of water, chemisorption of CO2 and cleavage of the C-O bond-the most energy consuming elementary steps in other catalysts such as copper-become nearly spontaneous at the W2C surface. This results in instantaneous formation of adsorbed CO-an important reaction intermediate-and an unlimited source of protons near the tungsten surface sites that are the main reasons for the observed superior activity, selectivity, and small potential.

Determination of pharmaceuticals in solid samples in municipal wastewater treatment plants by online SPE LC-MS/MS using QuEChERS extraction.

In this study, a pretreatment method based on the QuEChERS method has been applied for simultaneously extracting 27 residual pharmaceuticals from wastewater solids. The extracted compounds have been analyzed using online solid-phase extraction (SPE) coupled to liquid chromatography with tandem mass spectrometry (LC-MS/MS). A recovery test was conducted according to the absorbent type, and buffers were added in the sample extraction step. The highest recovery efficiency could be observed when Na2SO4 was used as an absorbent and Na2EDTA was injected during the extraction process; the recovery efficiencies of the proposed method for the target compounds ranged from 61.3 to 137.2%, and the repeatability was 6.8%. These recovery and repeatability data showed that the proposed method could reliably analyze the 27 target residual pharmaceuticals. The concentrations of the target compounds were all below the limits of quantification: 830 ng g-1 for the target compounds in suspended solids, 2353 ng g-1 in activated sludge, and 1929 ng g-1 in waste sludge. The analytical method established in this study can be applied to quantify residual pharmaceuticals in solid samples and to investigate their behaviors in a municipal wastewater treatment plant.

Spatial variation of pharmaceuticals in the unit processes of full-scale municipal wastewater treatment plants in Korea.

Several reports have elucidated the removal of pharmaceutical residues in municipal wastewater treatment plants (WWTPs). However, there remains a need to determine the spatial distribution of pharmaceuticals in the unit processes of full-scale municipal WWTPs. Herein, spatial variations of fifteen pharmaceuticals in the unit processes of four full-scale municipal WWTPs were assessed by analyzing both solid and liquid samples. Furthermore, different pathways of each pharmaceutical such as biodegradation, adsorption, deconjugation, and electrostatic interaction were investigated. Pharmaceutical mass loading were measured at various points for the different unit process and evaluated using liquid chromatography-tandem mass spectrometry. The average mass loading of acetaminophen and caffeine decreased tremendously in the first biological treatment process regardless of the process configuration. In contrast, a temporary increase was observed in the mass loading of ibuprofen in the anaerobic and/or anoxic processes, which was presumably caused by deconjugation. Additionally, the adverse effect of coagulation on ibuprofen removal was validated. The major removal mechanism for the selected antibiotics, except for sulfamethoxazole, was the adsorption by biosolids due to electrostatic interaction. Subsequently, a drastic decrease was observed in their mass loadings in the solid-liquid separation process of the WWTPs. The membrane bioreactor (MBR) shows excellent capability for mitigation of pharmaceuticals in municipal wastewater because it comprises a high concentration of biosolids that act as adsorbents. The evaluation of the spatial variations of the selected pharmaceuticals in different unit processes provides valuable information on their behavior and removal mechanisms.

Decreased Maximal Tongue Protrusion Length May Predict the Presence of Dysphagia in Stroke Patients.

OBJECTIVE: To investigate the relationship between maximal tongue protrusion length (MTPL) and dysphagia in post-stroke patients. METHODS: Free tongue length (FTL) was measured using the quick tongue-tie assessment tool and MTPL was measured using a transparent plastic ruler in 47 post-stroke patients. The MTPL-to-FTL (RMF) ratio was calculated. Swallowing function in all patients was evaluated via videofluoroscopic swallowing study (VFSS), PenetrationAspiration Scale (PAS), Functional Oral Intake Scale (FOIS), and Videofluoroscopic Dysphagia Scale (VDS). RESULTS: The MTPL and RMF values were significantly higher in the non-aspirator group than in the aspirator group (MTPL, p=0.0049; RMF, p<0.001). MTPL and RMF showed significant correlations with PAS, FOIS and VDS scores. The cut-off value in RMF for the prediction of aspiration was 1.56, with a sensitivity of 84% and a specificity of 86%. CONCLUSION: There is a relationship between MTPL and dysphagia in post-stroke patients. MTPL and RMF can be useful for detecting aspiration in post-stroke patients.

Distribution and Removal of Pharmaceuticals in Liquid and Solid Phases in the Unit Processes of Sewage Treatment Plants.

In this study, we analyzed 27 pharmaceuticals in liquid and solid phase samples collected from the unit processes of four different sewage treatment plants (STPs) to evaluate their distribution and behavior of the pharmaceuticals. The examination of the relative distributions of various categories of pharmaceuticals in the influent showed that non-steroidal anti-inflammatory drugs (NSAIDs) were the most dominant. While the relative distribution of antibiotics in the influent was not high (i.e., 3%-5%), it increased to 14%-30% in the effluent. In the four STPs, the mass load of the target pharmaceuticals was reduced by 88%-95% mainly in the biological treatment process, whereas the ratio of pharmaceuticals in waste sludge to those in the influent (w/w) was only 2%. In all the STPs, the removal efficiencies for the stimulant caffeine, NSAIDs (acetaminophen, naproxen, and acetylsalicylic acid), and the antibiotic cefradine were high; they were removed mainly by biological processes. Certain compounds, such as the NSAID ketoprofen, contrast agent iopromide, lipid regulator gemfibrozil, and antibiotic sulfamethoxazole, showed varying removal efficiencies depending on the contribution of biodegradation and sludge sorption. In addition, a quantitative meta-analysis was performed to compare the pharmaceutical removal efficiencies of the biological treatment processes in the four STPs, which were a membrane bioreactor (MBR) process, sequencing batch reactor (SBR) process, anaerobic-anoxic-oxic (A2O) process, and moving-bed biofilm reactor (MBBR) process. Among the biological processes, the removal efficiency was in the order of MBR > SBR > A2O > MBBR. Among the tertiary treatment processes investigated, powdered activated carbon showed the highest removal efficiency of 18%-63% for gemfibrozil, ibuprofen, ketoprofen, atenolol, cimetidine, and trimethoprim.

CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-ß (TGF-ß) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.

Osteopontin Protects Colonic Mucosa from Dextran Sodium Sulfate-Induced Acute Colitis in Mice by Regulating Junctional Distribution of Occludin.

BACKGROUND: Osteopontin (OPN) has been reported to play an important role in intestinal mucosal protection. Although OPN may have positive effects on tight junctions, the exact relationship between OPN and tight junctions has yet to be elucidated. AIMS: To investigate the role of OPN on tight junctions. METHODS: We evaluated clinical signs and histopathology of acute colitis induced by dextran sodium sulfate (DSS) in OPN knockout and wild-type (WT) mice in vivo. Expression levels of occludin and zonula occludens-1 were examined using immunofluorescence. For in vitro analysis, an siRNA-mediated OPN-suppressed Caco-2 monolayer was used. Expression levels and patterns of occludin were analyzed by immunofluorescence, and transepithelial electrical resistance (TER) was measured to evaluate barrier function. Triton X-100 fractionation was used to analyze phosphorylated occludin associated with tight junctional localization. RESULTS: OPN deficiency resulted in an elevated disease activity index, shortened colon length, and aggravated histological signs in mice with DSS-induced acute colitis compared to WT mice. OPN deficiency decreased occludin expression in the colonic mucosa. In Caco-2 monolayers, OPN suppression reduced junctional occludin and redistributed it into the intracellular compartment with decreased TER. Furthermore, western blot for occludin from Triton X-100 insoluble fraction revealed that OPN suppression reduced the phosphorylated form of occludin, which is actually distributed in the tight junction. CONCLUSIONS: Our study showed that OPN is essential for maintaining the tight junction complex by allowing occludin to localize at tight junctions. This could constitute additional evidence that OPN plays a crucial role in intestinal mucosal protection.

Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation.

AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders.

Membrane fouling control and enhanced removal of pharmaceuticals and personal care products by coagulation-MBR.

We investigated the effects of the addition of two coagulants-polyaluminium chloride (PACl) and chitosan-into the membrane bioreactor (MBR) process on membrane fouling and the removal of pharmaceuticals and personal care products (PPCPs). Their addition at optimized dosages improved the permeability of the membrane by reducing the concentration of soluble microbial products in mixed liquor, the content of inorganic elements, and irreversible fouling of the membrane surface. During long-term operation, the addition of PACl increased removal efficiencies of tetracycline, mefenamic acid, atenolol, furosemide, ketoprofen, and diclofenac by 17-23%. The comparative evaluation using mass balance calculations between coagulation-MBR (with PACl addition) and control-MBR (without PACl addition) showed that enhanced biodegradability played a key role in improving removal efficiencies of some PPCPs in coagulation-MBR. Coagulation-MBR also had higher oxygen uptake rates and specific nitrification rates of microorganisms. Overall, our findings suggest that the combination of MBR with coagulation reduced membrane fouling, lengthening operation period of the membrane, and improved the removal of some PPCPs as a result of enhanced biodegradability.

Removal of pharmaceuticals and personal care products by ammonia oxidizing bacteria acclimated in a membrane bioreactor: Contributions of cometabolism and endogenous respiration.

We carried out batch experiments using biomass from a membrane bioreactor (MBR) to study the influence of ammonia oxidizing bacteria (AOB) on the removal of 45 pharmaceuticals and personal care products (PPCPs). Kinetic parameters such as biodegradation constants and adsorption coefficients with and without AOB inhibition were estimated. No significant differences in adsorption tendency were found, but the biodegradability of most compounds was enhanced when ammonia was completely oxidized, indicating that AOB present in MBR played a critical role in eliminating the PPCPs. Moreover, target PPCPs were degraded in 2 stages, first by cometabolic degradation related to AOB growth, and then by endogenous respiration by microorganisms in the absence of other growth substrate. The compounds were classified into 3 groups according to removal performance and cometabolic degradation. Our approach provides new insight into the removal of PPCPs via cometabolism and endogenous respiration under AOB enrichment cultures developed in MBR.

Removal characteristics of pharmaceuticals and personal care products: Comparison between membrane bioreactor and various biological treatment processes.

We investigated the concentrations of 57 target compounds in the different treatment units of various biological treatment processes in South Korea, including modified biological nutrient removal (BNR), anaerobic-anoxic-aerobic (A2O), and membrane bioreactor (MBR) systems, to elucidate the occurrence and removal fates of PPCPs in WWTPs. Biological treatment processes appeared to be most effective in eliminating most PPCPs, whereas some PPCPs were additionally removed by post-treatment. With the exception of the MBR process, the A2O system was effective for PPCPs removal. As a result, removal mechanisms were evaluated by calculating the mass balances in A2O and a lab-scale MBR process. The comparative study demonstrated that biodegradation was largely responsible for the improved removal performance found in lab-scale MBR (e.g., in removing bezafibrate, ketoprofen, and atenolol). Triclocarban, ciprofloxacin, levofloxacin and tetracycline were adsorbed in large amounts to MBR sludge. Increased biodegradability was also observed in lab-scale MBR, despite the highly adsorbable characteristics. The enhanced biodegradation potential seen in the MBR process thus likely plays a key role in eliminating highly adsorbable compounds as well as non-degradable or persistent PPCPs in other biological treatment processes.

Suppression of osteopontin inhibits chemically induced hepatic carcinogenesis by induction of apoptosis in mice.

Previous clinical reports have found elevated osteopontin (OPN) levels in tumor tissues to be indicative of greater malignancy in human hepatocellular carcinoma (HCC). However, the role of OPN on carcinogenesis and its underlying mechanism remain unclear. In the present study, we investigated the oncogenic role of OPN in diethylnitrosamine (DEN)-induced hepatic carcinogenesis in mice. The overall incidence of hepatic tumors at 36 weeks was significantly lower in OPN knockout (KO) mice than in wild-type (WT) mice. Apoptosis was significantly enhanced in OPN KO mice, and was accompanied by the downregulation of epidermal growth factor receptor (EGFR). In the in vitro study, OPN suppression also led to lower mRNA and protein levels of EGFR associated with the downregulation of c-Jun in Hep3B and Huh7 human HCC cells lines, which resulted in increased apoptotic cell death in both cell lines. Moreover, a positive correlation was clearly identified between the expression of OPN and EGFR in human HCC tissues. These data demonstrate that the OPN deficiency reduced the incidence of chemically induced HCC by suppressing EGFR-mediated anti-apoptotic signaling. An important implication of our findings is that OPN positively contributes to hepatic carcinogenesis.

Control of membrane fouling with the addition of a nanoporous zeolite membrane fouling reducer to the submerged hollow fiber membrane bioreactor.

The membrane fouling control via the addition of nanoporous zeolite membrane fouling reducer (Z-MFR) to the submerged membrane bioreactor (MBR) was investigated. Using scanning electron microscopy/energy-dispersive X-ray (SEM/EDX) analysis techniques, the characteristics of fouling on a hollow fiber membrane surface were also analyzed. The addition of Z-MFR to the MBR led to the adsorption of foulants and the flocculation of mixed liquor suspended solids (MLSSs), which resulted in substantially enhancing the membrane filterability. The critical flux values obtained from the sewage mixed liquors of 3400 mg L(-1) at the effective dosage rate of 0.03 mg Z-MFR mg(-1) MLSS was 85 L m(-2) h(-1) (LMH), which was enhanced by 42%. The transmembrane pressure (TMP) variation under the operating conditions of 30 LMH with 3500 mg MLSS L(-1) showed that the addition of Z-MFR extended the time required to reach the critical flux of 0.32 bar by 2.6-fold longer than the control. Thus, due to the hybrid functions of adsorbing foulants and precipitating colloidal substances with the addition of Z-MFR, a decrease in the foulant amount and an improvement of sludge flocculation have been attained simultaneously. As a result, the membrane fouling control was achieved effectively with the addition of the Z-MFR.

Ablation of osteopontin suppresses N-methyl-N-nitrosourea and Helicobacter pylori-induced gastric cancer development in mice.

Several clinical studies have reported increased expression of osteopontin (OPN) in various types of human cancer, including gastric cancer. However, the precise mechanisms underlying tumor development remain unclear. In the present study, we investigated the pathogenic roles of OPN in Helicobacter pylori-induced gastric cancer development. Wild-type (WT) and OPN knockout (KO) mice were treated with N-methyl-N-nitrosourea (MNU) and infected with H.pylori. Mice were killed 50 weeks after treatment, and stomach tissues were assessed by histopathological examination, immunohistochemistry, quantitative real-time RT-PCR and western blotting. To clarify the carcinogenic effects of OPN, we also conducted an in vitro study using AGS human gastric cancer cell line and THP-1 human monocytic cell line. The overall incidence of gastric tumors was significantly decreased in OPN KO mice compared with WT mice. Apoptotic cell death was significantly enhanced in OPN KO mice and was accompanied by upregulation of signal transducer and activator of transcription 1 (STAT1) and inducible nitric oxide synthase (iNOS). In vitro study, OPN suppression also caused STAT1 upregulation and iNOS overexpression in AGS and THP-1 cells, which resulted in apoptosis of AGS cells. In addition, a negative correlation was clearly identified between expression of OPN and iNOS in human gastric cancer tissues. Our data demonstrate that loss of OPN decreases H.pylori-induced gastric carcinogenesis by suppressing proinflammatory immune response and augmenting STAT1 and iNOS-mediated apoptosis of gastric epithelial cells. An important implication of these findings is that OPN actually contributes to the development of gastric cancer.

Silencing of voltage-gated potassium channel KV9.3 inhibits proliferation in human colon and lung carcinoma cells.

Voltage-gated potassium (Kv) channels are known to be involved in cancer development and cancer cell proliferation. KV9.3, an electronically silent subunit, forms heterotetramers with KV2.1 in excitable cells and modulates its electrophysiological properties. However, the role of KV9.3 alone in non-excitable cancer cells has not been studied. Here, we evaluated the effect of silencing KV9.3 on cancer cell proliferation in HCT15 colon carcinoma cells and A549 lung adenocarcinoma cells. We confirmed the expression of KV9.3 mRNA in HCT15 and A549 cells and showed that silencing KV9.3 using small interfering RNA caused G0/G1 cell cycle arrest and alterations in cell cycle regulatory proteins in both HCT15 and A549 cells without affecting apoptosis. Also, stable knockdown of KV9.3 expression using short-hairpin RNA inhibited tumor growth in SCID mouse xenograft model. Using a bioinformatics approach, we identified Sp1 binding sites in the promoter region of the gene encoding KV9.3. We further found that Sp1 bound to this region and showed that the Sp1 inhibitor, mithramycin A, induced a concentration-dependent decrease in KV9.3 expression. Taken together, these data suggest that knockdown of KV9.3 inhibits proliferation in colon carcinoma and lung adenocarcinoma cell lines and may be regulated by Sp1.