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Purpose: In Alzheimer disease (AD), brain regions such as the cortex and the hippocampus show abundant amyloid load which correlates with cognitive function decline. Prior to the significant development of AD pathophysiology, patients report the manifestation of neuropsychiatric symptoms, indicating a functional interplay between basal ganglia structures and hippocampal regions. Zinc finger and BTB domain-containing protein 16 (ZBTB16) is a transcription factor that controls the expression of downstream genes and the involvement of ZBTB16 in the striatum undergoing pathological aging in AD and the resulting behavioral phenotypes has not yet been explored.Methods: To study molecular alterations in AD pathogenesis, we analyzed the brain from amyloid precursor protein (APP)/ presenilin 1 (PS1) transgenic mice. The molecular changes in the striatal region of the brain were analyzed via the immunoblotting, and the quantitative RNA sequencing. The cognitive impairments of APP/PS1 mice were assessed via 3 behavioral tests: 3-chamber test, Y-maze test, and noble object recognition test. And multielectrode array experiments for the analysis of the neuronal activity of the striatum in APP/PS1 mice was performed.Results: We found that the alteration in ZBTB16 levels that occurred in the early ages of the pathologically aging striatum coalesces with the disruption of transcriptional dysregulation while causing social memory deficits, anxiety-like behavior. The early ZBTB16 knockdown treatment in the striatum of APP/PS1 mice rescued cognition that continued into later age.Conclusions: This study demonstrates that perturbation of transcriptional regulation of ZBTB16 during pathological aging may influence cognitive impairments and reveals a potent approach to targeting the transcriptional regulation of the striatum for the treatment of AD.

CD45RA+ effector memory (EM) CD8+ T cell expansion was reported in Alzheimer’s disease (AD). Such cells are IL-7 receptor alpha (IL-7Rα)low EM CD8+ T cells, which expand with age and have a unique aging gene signature (i.e., IL-7Rαlow aging genes). Here we investigated whether IL-7Rαlow aging genes and previously reported AD and memory (ADM) genes overlapped with clinical significance in AD patients. RT-qPCR analysis of 40 genes, including 29 ADM, 9 top IL-7Ralow aging and 2 control genes, showed 8 differentially expressed genes between AD and cognitively normal groups; five (62.5%) of which were top IL-7Rαlow aging genes. Over-representation analysis revealed that these genes were highly present in molecular and biological pathways associated with AD. Distinct expression levels of these genes were associated with neuropsychological testing performance in 3 subgroups of dementia participants. Our findings support the possible implication of the IL-7Rαlow aging gene signature with AD.

Safety and toxicity in mouse after treatment of linalool with different dose.

Purpose: Bromodomain-containing protein 7 (BRD7), which is a subunit of SWI/SNF complex, has been recently suggested as a novel tumor suppressor in several cancers. In this study, we investigated the tumor suppressive effect of BRD7 in epithelial ovarian cancer.Experimental Design: We analyzed the expression of BRD7 in human ovarian tissues with real-time PCR. To investigate the functional role of BRD7, we transfected ovarian cancer cells (A2780 and SKOV3) with BRD7 plasmid and checked the cell viability, apoptosis, and invasion. The activities of BRD7 in the signaling pathways associated with carcinogenesis were also tested. In addition, we used the orthotopic mouse model for ovarian cancer to evaluate tumor growth-inhibiting effect by administration of BRD7 plasmid.Results: The BRD7 expression was downregulated in the ovarian cancer tissues compared with normal (P < 0.05), high-grade serous cancer exhibited significantly decreased expression of BRD7 compared with low-grade (P < 0.01) serous cancer. Transfection of BRD7 plasmid to A2780 (p53-wild) or SKOV3 (p53-null) ovarian cancer cells showed the tumor suppressive effects assessed by cell viability, apoptosis, and invasion assay and especially significantly decreased tumor weight in orthotopic mouse model (A2780). Moreover, we found that tumor suppressive effects of BRD7 are independent to the presence of p53 activity in ovarian cancer cells. BRD7 negatively regulated β-catenin pathway, resulting in decreased its accumulation in the nucleus.Conclusions: These results suggested that BRD7 acts as a tumor suppressor in epithelial ovarian cancers independently of p53 activity, via negative regulation of β-catenin pathway. Clin Cancer Res; 20(3); 565–75. ©2013 AACR.

Purpose:Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity.Experimental Design: Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues.Results:Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (81.8% reduction in SKOV3ip1, P = 0.017; 84.3% reduction in HeyA8, P < 0.005). These effects were mediated by decreased tumor cell proliferation and angiogenesis, and increased tumor cell apoptosis. Next, we assessed the effects of Src silencing on other SFK members in ovarian cancer cell lines. Src silencing resulted in significantly increased Fgr levels. Dual Src and Fgr silencing in vitro resulted in increased apoptosis that was mediated by increased caspase and AKT activity. In addition, dual silencing of Src and Fgr in vivo using siRNA/CH-NP resulted in the greatest reduction in tumor growth compared with silencing of either Src or Fgr alone in the HeyA8 model (68.8%, P < 0.05).Conclusions: This study demonstrates that, in addition to Src, Fgr plays a biologically significant role in ovarian cancer growth and might represent an important target. Clin Cancer Res; 17(7); 1713–21. ©2011 AACR.

PDF - 1246K, Western blot analysis was used to determine expression levels of MEK1 and p-ERK (1/2) when two ovarian cancer cells were transfected with BRD7 plasmid.

Supplementary Data from EphA2 Targeted Chemotherapy Using an Antibody Drug Conjugate in Endometrial Carcinoma

Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA).Experimental Design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with ανβ3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma.Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the ανβ3 integrin–positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls.Conclusions: This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease. Clin Cancer Res; 16(15); 3910–22. ©2010 AACR.

Supplementary Figures S1-S5.

Supplementary Data from Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

Supplementary Data from c-Jun-NH2-kinase-1 Inhibition Leads to Antitumor Activity in Ovarian Cancer

PDF - 57K, Information of the cell lines used in experiments.

Purpose: EphA2 overexpression is frequently observed in endometrial cancers and is predictive of poor clinical outcome. Here, we use an antibody drug conjugate (MEDI-547) composed of a fully human monoclonal antibody against both human and murine EphA2 (1C1) and the tubulin polymerization inhibitor monomethylauristatin F.Experimental Design: EphA2 expression was examined in endometrial cancer cell lines by Western blot. Specificity of MEDI-547 was examined by antibody degradation and internalization assays. Viability and apoptosis were investigated in endometrial cancer cell lines and orthotopic tumor models.Results: EphA2 was expressed in the Hec-1A and Ishikawa cells but was absent in the SPEC-2 cells. Antibody degradation and internalization assays showed that the antibody drug conjugate decreased EphA2 protein levels and was internalized in EphA2-positive cells (Hec-1A and Ishikawa). Moreover, in vitro cytotoxicity and apoptosis assays showed that the antibody drug conjugate decreased viability and increased apoptosis of Hec-1A and Ishikawa cells. In vivo therapy experiments in mouse orthotopic models with this antibody drug conjugate resulted in 86% to 88% growth inhibition (P < 0.001) in the orthotopic Hec-1A and Ishikawa models compared with controls. Moreover, the mice treated with this antibody drug conjugate had a lower incidence of distant metastasis compared with controls. The antitumor effects of the therapy were related to decreased proliferation and increased apoptosis of tumor and associated endothelial cells.Conclusions: The preclinical data for endometrial cancer treatment using MEDI-547 show substantial antitumor activity. Clin Cancer Res; 16(9); 2562–70. ©2010 AACR.

Purpose: To show the functional, clinical, and biological significance of c-Jun-NH2-kinase (JNK)-1 in ovarian carcinoma.Experimental Design: Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of N-5-[4-(4-methyl piperazine methyl)-benzoylamido]-2-methylphenyl-4-[3-(4-methyl)-pyridyl]-2-pyrimidine amine (WBZ_4), a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer.Results: We found a significant association of pJNK with progression-free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose-dependent fashion in four ovarian cancer cell lines. In vivo, whereas imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The antitumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights compared with nonsilencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition.Conclusions: These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the redesigned WBZ_4 compound should be considered for further clinical development. Clin Cancer Res; 16(1); 184–94

PDF - 1244K, BRD7 mediated reduction of MMP2 require beta-catenin pathway. A2780 (A) or SKOV3 (B) cells were co-transfected with BRD7 plasmid and beta-catenin siRNA. After 36 hours, whole mRNA was isolated and cDNA was synthesized. Semi-quantitative PCR was performed to estimate the expression of MMP2 mRNA.

PDF - 1513K, BRD7 mediated tumor suppressive effects are not correlated with cell cycle. A, Cell cycle distributions of A2780 (marked as A) and SKOV3 (marked as S) cells measured by flow cytometry. Cell number histograms plot (upper panel) show the distribution of cells on the basis of DNA contents as determined by PI fluorescence intensity. Left peak represents cells in the G1/G0 phases, hatched area represents cells in S phase, and peak on right represents cells in the G2 and M phases. The expression levels of BRD7 mRNA among phases of cell cycle show no significant differences in both A2780 and SKOV3 cell line (.lower panel). Values are expressed as means of three independent experiments plus or minus SD. B, Western blot analysis was used to determine expression levels of Cyclin B1, D1 or E2.

PDF - 856K, Western blot analysis was used to determine expression levels of alpha-catenin when two ovarian cancer cells were transfected with BRD7 plasmid.

Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth.Experimental Design: Expression of dopamine receptors (DR1–DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP).Results: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely blocked with daily dopamine treatment. Dopamine treatment also blocked the stress-induced increase in angiogenesis. Endothelial and ovarian cancer cells expressed all dopamine receptors except for the lack of DR3 expression in ovarian cancer cells. DR2 was responsible for the inhibitory effects of dopamine on tumor growth and microvessel density as well as the stimulatory effect on apoptosis, as the DR2 antagonist eticlopride reversed these effects. Dopamine significantly inhibited cell viability and stimulated apoptosis in vitro. Moreover, dopamine reduced cyclic AMP levels and inhibited norepinephrine and vascular permeability factor/VEGF-induced Src kinase activation.Conclusions: Dopamine depletion under chronic stress conditions creates a permissive microenvironment for tumor growth that can be reversed by dopamine replacement. Clin Cancer Res; 17(11); 3649–59. ©2011 AACR.

Radiofrequency radiation (RFR) was classified as a "possible" human carcinogen in 2011, which caused great public concern. A carcinogenicity study by the National Toxicology Program (NTP) found Code Division Multiple Access-and Global System for Mobile Communications-modulated mobile phone RFR to be carcinogenic to the brain and heart of male rats. As part of an investigation of mobile phone carcinogenesis, and to verify the NTP study results, a 5-year collaborative animal project was started in Korea and Japan in 2019. An international animal study of this type has two prerequisites: use of the same study protocol and the same RF-exposure system. This article discusses our experience in the design of this global study on radiofrequency electromagnetic fields (RF-EMFs).© 2022 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

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.

INTRODUCTIONEffector memory (EM) CD8+ T cells have been associated with poor cognition in Alzheimer’s disease (AD). Our lab recently discovered an age-associated gene expression signature of IL-7 receptor alpha (IL-7Rα)low EM CD8+ T cells. We hypothesized that individuals with AD have altered levels of this IL-7Rαlow aging gene expression.METHODSForty genes associated with IL-7Rαlow EM CD8+ T cells, AD, or memory, were analyzed in peripheral blood of participants with normal cognition, mild cognitive impairment, and dementia by qPCR.RESULTSOf the eight genes that were found to be differentially expressed based on clinical diagnosis, 5 genes (62.5%) were IL-7Rαlow aging genes. Principal component analysis revealed 3 clusters of participants with dementia which had distinct expression levels of IL-7Rαlow aging genes and cognitive function.DISCUSSIONOur findings support the possible relationship of the IL-7Rαlow EM CD8+ T cell aging signature with cognition in individuals with dementia due to AD.

BackgroundAducanumab (Adu), which is a human IgG1 monoclonal antibody that targets oligomer and fibril forms of beta-amyloid (Aβ), has been reported to reduce amyloid pathology and improve impaired cognition after the administration of a high dose (10 mg/kg) of the drug in Alzheimer’s disease (AD) clinical trials. The purpose of this study is to investigate the effects of a lower dose of Adu (3mg/kg) with enhanced delivery via focused ultrasound (FUS) in an AD mouse model.MethodsThe FUS with microbubbles opened the blood-brain barrier of the hippocampus for the delivery of Aducanumab. Combined therapy of FUS and Aducanumab was performed three times in total and each treatment was performed biweekly. Y-maze test, Brdu labeling, 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.ResultsThe combined treatment with FUS markedly increased the delivery of Adu into the brain by approximately 8.1 times in the brains. The combined treatment significantly restored cognitive impairment and decreased the level of amyloid plaques in the hippocampi of 5xFAD mice compared with Adu or FUS alone. The combined treatment with FUS and Adu increased reactive microglia and astrocytes associated with amyloid plaques in the hippocampi of 5xFAD mice. Furthermore, RNA sequencing identified 4 enriched canonical pathways such as phagosome formation, neuroinflammation signaling, CREB signaling and reelin signaling was altered in the hippocami of 5xFAD given the combined treatment. ConclusionIn conclusion, the enhanced delivery of a low dose of Aducanumab via FUS decreased amyloid deposits and restored cognitive function. This study provides better insight into establishing a solid therapeutic strategy for the treatment of AD as well as other neurodegenerative diseases.

Background: The diverse roles of ubiquitously present translationally controlled tumor protein (TCTP) have been well delineated in several organs, but its possible function in the brain, especially with regard to memory function, has not received much attention. This study describes the effects of TCTP on mice with memory impaired by scopolamine (SCO) administration. Specifically, the memory and synaptic functions of 7- and 12-month-old SCO-treated wild mice (WT) were compared with those of TCTP-overexpressing (TG) and TCTP knocked down (KD) mice. Methods: Passive-avoidance tasks were performed on WT, TG and KD mice for 4 weeks after intraperitoneal injection with SCO (1 mg/kg) or saline (CON). After completion of the behavioral studies, the hippocampi were collected and their PSD-95, synapsin-1 and synaptophysin contents analyzed by western blocking and immunohistochemical analyses, and compared with those of 5xfamilial Alzheimer's disease (5xFAD) mice and postmortem AD patients. Results: The SCO-induced memory impairment was restored in TCTP-TG to that of WT level, but not in KD. Hippocampal expression of PSD-95, synapsin-1, and synaptophysin was increased in TG-SCO but decreased in KD-SCO mice. The decreased levels of TCTP, PSD-95, and synaptophysin were also found in the hippocampi of 5xFAD mice and AD patients. PSD-95 immunoreactivity increased particularly in dentate gyrus and CA1 in TCTP-TG, but reduced in KD. The p-CREB/CREB and brain-derived neurotrophic factor (BDNF) expressions also increased in TCTP-TG but dramatically decreased in KD. Conclusion: TCTP restores damaged memory in mice possibly by increasing synaptic function. Keywords TCTP, transgenic, knockdown, scopolamine-induced dementia, memory, synaptic function, PSD-95, synaptophysin, synapsin-1, CREB, BDNF

Astrocytes are the most abundant cell type in the central nervous system (CNS) and their major function is to maintain homeostasis of the CNS by exerting various functions. Simultaneously, reactive astrocytes are well known to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD). Reactive astrocytes, induced by amyloid beta peptide (Aβ), the main component of the neuritic plaques found in AD, induce neuroinflammation, producing cytokines that lead to neuronal cell death in AD. Phloroglucinol,a polyphenol monomer and a component of phlorotannin, is found at sufficient levels in Ecklonia cava of the Laminariaceae family. Recently, several studies have reported that phloroglucinol has the ability to trap free radicals in lung fibroblasts or cancer cells. However, the effects of phloroglucinol in astrocytes have not yet been studied. Here, we found that phloroglucinol inhibits the generation of ROS induced by oligomeric Aβ1-42 (oAβ1-42) treatment in primary astrocytes. Futhermore, phloroglucinol was shown to ameliorate the protein expression of glial fibrillary acidic protein, a marker of reactive astrocytes, after treatment with oAβ1-42. These results indicate that phloroglucinol exerts antioxidant effects in primary cultured astrocytes and attenuates the astrocytic activation induced by oAβ1-42.

The placenta protects the fetus against excessive stress-associated maternal cortisol during pregnancy. We studied whether exposure to radiofrequency electromagnetic field (RF-EMF) radiation during pregnancy can cause changes in dams and their placentas. Pregnant Sprague-Dawley rats were divided into cage-control, sham-exposed, and RF-exposed groups. They were exposed to RF-EMF signals at a whole-body specific absorption rate of 4 W/kg for 8 h/day from gestational Day 1 to 19. Levels of cortisol in the blood, adrenal gland, and placenta were measured by enzyme-linked immunosorbent assay. Levels of adrenocorticotropic hormone and corticotropin-releasing hormone were monitored in maternal blood. Expression levels of placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) messenger RNA (mRNA) were measured by reverse transcription polymerase chain reaction. Morphological changes in the placenta were analyzed using hematoxylin and eosin staining. Fetal parts of the placenta were measured using Zen 2.3 blue edition software. Maternal cortisol in circulating blood (RF: 230 ± 24.6 ng/ml and Sham: 156 ± 8.3 ng/ml) and the adrenal gland (RF: 58.3 ± 4.5 ng/ml and Sham: 30 ± 3.8 ng/ml) was significantly increased in the RF-exposed group (P < 0.05). Placental cortisol was stably maintained, and the level of placental 11β-HSD2 mRNA expression was not changed in the RF-exposed group. RF-EMF exposure during pregnancy caused a significant elevation of cortisol levels in circulating blood; however, no changes in the placental barrier were observed in pregnant rats. Bioelectromagnetics. © 2021 Bioelectromagnetics Society.