Integrative analysis of circular RNA regulatory network in papillary thyroid carcinoma.

Papillary thyroid cancer (PTC) is the most common type of endocrine cancer worldwide. Generally, PTC has an excellent prognosis; however, lymph node metastases and recurrences occur frequently. Over the last decade, circular RNAs (circRNAs), a large class of noncoding RNAs (ncRNAs), have emerged as key regulators of various tumor progression pathways. Here, we aimed to identify novel circRNAs as PTC biomarkers. Differentially expressed circRNAs and mRNAs were analyzed using public datasets from the Gene Expression Omnibus and Cancer Genome Atlas. In addition, we screened for target miRNAs using online prediction databases. Based on these results, we established a circRNA-miRNA-mRNA regulatory network associated with PTC, in which protein-protein interaction networks led to the identification of hub genes. Functional enrichment and survival analyses were performed to gain insights into the biological mechanisms of circRNA involvement. As a result, we found that two circRNAs (hsa_circ_0041829 and has_circ_0092299), four miRNAs (miR-369, miR-486, miR-574, and miR-665), and nine hub genes (BBC3, E2F1, FYN, MAG, SDC1, SDC3, SNAP25, TK1, and TYMS) play significant roles in PTC progression. This study provides a novel framework for understanding the roles of circRNA-miRNA-mediated gene regulation in PTC. It also introduces potential therapeutic targets and prognostic biomarkers, which may serve as a basis for developing targeted therapeutic interventions for PTC.

RUNX3 regulates the susceptibility against EGFR-targeted non-small cell lung cancer therapy using 47Sc-conjugated cetuximab.

BACKGROUND: Radioimmunotherapy with cetuximab and conjugates with various radioisotopes is a feasible treatment option for different tumor models. Scandium-47 (47Sc), one of several ß--particle-emitting radioisotopes, displays favorable physical and chemical properties for conjugation to monoclonal antibodies. However, the therapeutic efficacy of 47Sc in preclinical and clinical studies is largely unknown. Given that intrinsic alterations in tumors greatly contribute to resistance to anti-epidermal growth factor receptor (EGFR)-targeted therapy, research on overcoming resistance to radioimmunotherapy using cetuximab is required. METHODS: 47Sc was produced by irradiation of a CaCO3 target at the HANARO research reactor in KAERI (Korea Atomic Energy Research Institute) and prepared by chromatographic separation of the irradiated target. Cetuximab was conjugated with 47Sc using the bifunctional chelating agent DTPA. Radiochemical purity was determined using instant thin-layer chromatography. The immunoreactivity of 47Sc-DTPA-cetuximab was evaluated using the Lindmo method and an in vitro cell-binding assay. The inhibitory effects of cetuximab and 47Sc-DTPA-cetuximab were confirmed using cell growth inhibition and BrdU cell proliferation assays. Differences in protein expression levels between cetuximab- and 47Sc-DTPA-cetuximab-treated cells were confirmed using western blotting. Complex formation between RUNX3 and DNA repair components was confirmed using immunoprecipitation and western blotting. RESULTS: Cetuximab induces cell cycle arrest and cell death in EGFR-overexpressing NSCLC cells. Radiolabeling of cetuximab with 47Sc led to increased therapeutic efficacy relative to cetuximab alone. Application of 47Sc-DTPA-cetuximab induced DNA damage responses, and activation of RUNX3 significantly enhanced the therapeutic efficacy of 47Sc-DTPA-cetuximab. RUNX3 mediated susceptibility to EGFR-targeted NSCLC therapy using 47Sc-DTPA-cetuximab via interaction with components of the DNA damage and repair machinery. CONCLUSIONS: 47Sc-DTPA-cetuximab promoted cell death in EGFR-overexpressing NSCLC cells by targeting EGFR and inducing DNA damage as a result of ß irradiation emitted from the conjugated 47Sc. Activation of RUNX3 played a key role in DNA damage and repair processes in response to the ionizing radiation and inhibited cell growth, thus leading to more effective tumor suppression. RUNX3 can potentially moderate susceptibility to 47Sc-conjugated cetuximab by modulating DNA damage and repair process mechanisms.

A novel therapeutic anti­CD55 monoclonal antibody inhibits the proliferation and metastasis of colorectal cancer cells.

In recent years, efforts to treat cancer by improving the immune function of patients have received a great deal of attention. As part of the immune system, complement is also under such evaluation. Among the many components of the complement system, complement decay accelerating factor (CD55 or DAF) is known to inhibit complement­mediated cell lysis. However, little is known about the role of CD55 in terms of cancer therapy. The present study aimed to demonstrate that increased levels of CD55 are strongly correlated with the progression of colorectal cancer. A novel CD55 chimeric monoclonal antibody was developed that may boost the immune response, thereby suppressing cancer. The CD55 antibody treatment activated complement and therefore suppressed the proliferation, invasion and migration of colorectal cancer cells. This tumoricidal activity is partly explained by the inflammatory response via the activation of proinflammatory cytokines. In addition, the CD55 antibody treatment synergistically enhanced the tumoricidal activity of 5­FU in colorectal cancer cells, suggesting that combined treatment may be a better strategy in colorectal cancer therapy.

Ultrasound-enhanced scintillation proximity assay for rapid diagnostics.

Scintillation proximity assay (SPA) is a type of radioimmunoassay (RIA). We apply ultrasound enhancement to the general SPA. All assay procedures, including the antibody coating and radiolabeled antigen binding are achieved by simply mixing then standing for 5 min in an ultrasound chamber. No additional incubation time is required. To further demonstrate the capability of the UE-SPA, a quantitative measurement of CD55 in various grades of colon tumors was assessed on human tissue slides. The results showed a significant correlation between CD55 expression and tumorigenesis. In conclusion, we confirmed that UE-SPA is a reliable, rapid and alternative to RIA.

Development of a radionuclide-labeled monoclonal anti-CD55 antibody with theranostic potential in pleural metastatic lung cancer.

Decay-accelerating factor (CD55 or DAF) inhibits complement-dependent cytotoxicity. We determined that CD55 is overexpressed in 76.47% of human non-small cell lung cancer tissue specimens. We therefore developed a lutetium-177-labeled chimeric monoclonal antibody against CD55. CD55-specific single-chain variable fragment (scFv) was selected from a naïve chicken scFv phage-display library, converted to IgG, and radiolabeled with lutetium-177 to generate a 177Lu-anti-CD55 antibody. We then charaterized the biodistribution of this antibody in a mouse model of pleural metastatic lung cancer. The 177Lu-anti-CD55 antibody was primarily retained in tumor tissue rather than normal tissue. Treatment of the mice with 177Lu-anti-CD55 reduced the growth of lung tumors and improved median survival in vivo by two-fold compared to controls. Finally, 177Lu-anti-CD55 also enhanced the antitumor activity of cisplatin both in vitro and in vivo. These data suggest 177Lu-anti-CD55 antibody is a promising theranostic agent for pleural metastatic lung cancer.

Bcl-xL deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed.

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.

Beyond the Role of CD55 as a Complement Component.

The complement is a part of the immune system that plays several roles in removing pathogens. Despite the importance of the complement system, the exact role of each component has been overlooked because the complement system was thought to be a nonspecific humoral immune mechanism that worked against pathogens. Decay-accelerating factor (DAF or CD55) is a known inhibitor of the complement system and has recently attracted substantial attention due to its role in various diseases, such as cancer, protein-losing enteropathy, and malaria. Some protein-losing enteropathy cases are caused by CD55 deficiency, which leads to complement hyperactivation, malabsorption, and angiopathic thrombosis. In addition, CD55 has been reported to be an essential host receptor for infection by the malaria parasite. Moreover, CD55 is a ligand of the seven-span transmembrane receptor CD97. Since CD55 is present in various cells, the functional role of CD55 has been expanded by showing that CD55 is associated with a variety of diseases, including cancer, malaria, protein-losing enteropathy, paroxysmal nocturnal hemoglobinuria, and autoimmune diseases. This review summarizes the current understanding of CD55 and the role of CD55 in these diseases. It also provides insight into the development of novel drugs for the diagnosis and treatment of diseases associated with CD55.

Analyses of Ferrous and Ferric State in DynabiTab Using Mössbauer Spectroscopy.

Antianemic medicament ferrous gluconate, ferrous fumarate, and a Dynabi tablet with a basic iron bearing ingredient were studied with the use of Mössbauer spectroscopy. Room temperature spectra of ferrous gluconate gave clear evidence that the two phases of iron were present: ferrous (Fe2+) as a major one with a contribution at and above 91 a.u.% and ferric (Fe3+) whose contribution was found to be ~9 a.u.%. In the case of ferrous fumarate, a single phase was measured corresponding to ferrous (Fe2+) state. A Dynabi tablet consists of ferrous fumarate and ferrous fumarate. The ferric phase in ferrous gluconate is able to be reached about ~3.6 a.u.% in a tablet.

STAT5A-mediated NOX5-L expression promotes the proliferation and metastasis of breast cancer cells.

NADPH oxidase (NOX) generates reactive oxygen species (ROS) and has been suggested to mediate cell proliferation in some cancers. Here, we show that an increase in the expression of NOX5 long form (NOX5-L) is critical for tumor progression in breast tumor tissues. Immunostaining of clinical samples indicated that NOX5 was overexpressed in 41.1% of breast ductal carcinoma samples. NOX5-L depletion consistently suppressed cell proliferation, invasion, and migration in vitro. Antibody-mediated neutralization of NOX5-L attenuated tumor progression in a mouse xenograft model. Promoter analysis revealed that NOX5-L expression is regulated by STAT5A in breast cancer cells. Based on our novel findings, we suggest that inhibition of NOX5-L may be a promising therapeutic strategy that exerts anti-cancer effects via the modulation of ROS-mediated cell signaling.

An assessment tumor targeting ability of (177)Lu labeled cyclic CCK analogue peptide by binding with cholecystokinin receptor.

The cholecystokinin (CCK) receptor is known as a receptor that is overexpressed in many human tumors. The present study was designed to investigate the targeting ability of cyclic CCK analogue in AR42J pancreatic cells. The CCK analogues, DOTA-K(glucose)-Gly-Trp-Nle-Asp-Phe (DOTA-glucose-CCK) and DOTA-Nle-cyclo(Glu-Trp-Nle-Asp-Phe-Lys-NH2) (DOTA-[Nle]-cCCK), were synthesized and radiolabeled with (177)Lu, and competitive binding was evaluated. The binding appearance of synthesized peptide with AR42J cells was evaluated by confocal microscopy. And bio-distribution was performed in AR42J xenografted mice. Synthesized peptides were prepared by a solid phase synthesis method, and their purity was over 98%. DOTA is the chelating agent for (177)Lu-labeling, in which the peptides were radiolabeled with (177)Lu by a high radiolabeling yield. A competitive displacement of (125)I-CCK8 on the AR42J cells revealed that the 50% inhibitory concentration value (IC50) was 12.3 nM of DOTA-glucose-CCK and 1.7 nM of DOTA-[Nle]-cCCK. Radio-labeled peptides were accumulated in AR42J tumor in vivo, and %ID/g of the tumor was 0.4 and 0.9 at 2 h p.i. It was concluded that (177)Lu-DOTA-[Nle]-cCCK has higher binding affinity than (177)Lu-DOTA-glucose-CCK and can be a potential candidate as a targeting modality for a CCK receptor over-expressing tumors.

NOX5-L can stimulate proliferation and apoptosis depending on its levels and cellular context, determining cancer cell susceptibility to cisplatin.

The NADPH oxidase, NOX5, is known to stimulate cell proliferation in some cancers by generating reactive oxygen species (ROS). We show here that the long form of NOX5 (NOX5-L) also promotes cell death, and thus determines the balance of proliferation and death, in skin, breast and lung cancer cells. Moderate expression of NOX5-L induced cell proliferation accompanied by AKT and ERK phosphorylation, whereas an increase in NOX5-L above a certain threshold promoted cancer cell death accompanied by caspase-3 activation. Notably, cisplatin treatment increased NOX5-L levels through CREB activation and enhanced NOX5-L activity through augmentation of Ca2+ release and c-Abl expression, ultimately triggering ROS-mediated cancer cell death-a distinct pathway absent in normal cells. These results indicate that NOX5-L determines cellular responses in a concentration- and context-dependent manner.

Biological evaluation of (177)Lu-labeled DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 for gastrin-releasing peptide receptor-positive prostate tumor targeting.

Bombesin binds with selectivity and high affinity to a Gastrin-releasing peptide receptor (GRPR), which is highly overexpressed in prostate cancer cells. The present study describes the in vitro and in vivo biological characteristics of DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 (DOTA-sBBNA), an antagonist analogue of bombesin peptide for the targeting of GRPR. DOTA-sBBNA was synthesized and labeled with (177)Lu as previously published. A saturation assay on PC-3 human prostate cancer cells revealed that the Kd value of the radiolabeled peptide was 1.88 nM with a maximum binding capacity (Bmax) of 289.3 fmol/10(6) cells. The radio-peptide slowly internalized, and 24.4±0.5% of the total binding was internalized in 4hr. Biodistribution studies were conducted in healthy and PC-3 xenografted balb/c mice, which showed high uptake and retention of tumor-associated radioactivity in PC-3 xenografted mice. The tumor-to-blood ratio was 126.02±9.36 at 1.5hr p.i., and was increased to 216.33±61.58 at 24hr p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and rapidly cleared from the blood pool. The GRPR is also over-expressed in Korean prostate cancer patients. These results suggest that this (177)Lu-labeled peptide has promising characteristics for application in nuclear medicine, namely for the diagnosis and treatment of GRPR over-expressing prostate tumors.

Preclinical pharmacokinetic, biodistribution, imaging and therapeutic efficacy of (177)Lu-Labeled glycated bombesin analogue for gastrin-releasing peptide receptor-positive prostate tumor targeting.

UNLABELLED: The gastrin-releasing peptide receptor (GRPR) has been shown to be overexpressed in many human tumors, including prostate, colon, gastric, breast, pancreatic, and small cell lung cancers. Because bombesin (BBS) binds to GRPR with high affinity, BBS derivatives have been labeled with various radionuclides and have been demonstrated to be successful candidates for peptide receptor radiotherapy (PRRT). The present study describes the in vitro and in vivo preclinical characteristics of (177)Lu-DOTA-Lys(glucose)-4 aminobenzoic acid-BBS7-14 ((177)Lu-DOTA-gluBBN) to prepare radiolabeled candidates for the treatment of GRPR-expressing prostate tumors. METHODS: (177)Lu-DOTA-gluBBN was prepared as previously published [1]. Human prostate PC-3 tumor cells were used to determine the binding (Kd) retention and efflux of (177)Lu-DOTA-gluBBN. Pharmacokinetic, imaging, and radiotherapy studies were performed in PC-3 xenografted mice. RESULTS: The Kd value of (177)Lu-DOTA-gluBBN was 0.63 nM, with a maximum binding capacity (Bmax) of 669.7 fmol/10(6) cells (4.04×10(5) GRPR/cell). During a 2-hr incubation, 90.1±0.4% of the cell-associated radio-peptide was internalized, and 56.3±7.1% of the internalized radio-peptide was externalized in vitro. High amounts of the radio-peptide were rapidly accumulated in a PC-3 tumor in vivo, and the % ID/g of the tumor was 12.42±2.15 1 hr p.i. The radio-peptide was quickly cleared from the blood, yielding tumor-to-blood ratios of 39.22±17.36 at 1 hr p.i. and 330.67±131.23 at 24hr p.i. In addition, (177)Lu-DOTA-gluBBN was clearly visualized in PC-3 tumors 1 hr p.i. and significantly inhibited the tumor growth (P<0.05). Treatment-related toxicity in the pancreas and kidneys was not observed, except for slight glomerulopathy. CONCLUSIONS: The pharmacokinetic, imaging, and therapy studies suggest that this (177)Lu-DOTA-gluBBN has promising characteristics for application in nuclear medicine, namely, for the diagnosis and treatment of GRPR-overexpressing prostate tumors.

Synthesis and biological evaluation of a novel (177)Lu-DOTA-[Gly(3)-cyclized(Dap(4), (d)-Phe(7), Asp(10))-Arg(11)]α-MSH(3-13) analogue for melanocortin-1 receptor-positive tumor targeting.

In this study, a novel α-melanocyte stimulating hormone (α-MSH) analogue 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupled [Gly(3)-cyclized(Dap(4), (d)-Phe(7), Asp(10))-Arg(11)]α-MSH(3-13) (DOTA-GMSH) for melanocortin-1 receptor (MC-1R) targeting was newly synthesized, radiolabeled with (177)Lu, and in vitro and in vivo characterized. (177)Lu-labeled peptides were prepared with a high radiolabeling yield (>98%), and its Log p value was -2.89. No degradation was observed not only by serum incubation at 37°C for 7 days but also by an HPLC analysis of radioactive metabolites in urine. A cell binding assay revealed that an inhibitory concentration of 50% (IC(50)) of the peptide was 3.80 nM. The tumor-to-blood ratio, which was 14.27 at 2 hours p.i., was increased to 56.37 at 24 hours p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and was rapidly cleared from the blood pool. We, therefore, conclude that (177)Lu-DOTA-GMSH has promising characteristics for application in nuclear medicine, namely for the diagnosis of MC-1R over-expressing tumors.

Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retinal pigment epithelial cells.

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.

Cannabinoid receptor 1 mediates high glucose-induced apoptosis via endoplasmic reticulum stress in primary cultured rat mesangial cells.

The endocannabinoid system in animals and humans is involved in the onset of diverse diseases, including obesity and diabetic nephropathy, which is a major end-stage renal disease characterized by high glucose (HG)-induced apoptosis of mesangial cells. Endocannabinoids induce physiological and behavioral effects by activating two specific receptors, cannabinoid receptor 1 (CB(1)R) and cannabinoid receptor 2 (CB(2)R). However, the pathophysiology of CB(1)R in diabetic nephropathy has not been elucidated. We investigated the effects of HG on CB(1)R expression and its signaling pathways in primary cultured rat mesangial cells. HG significantly increased CB(1)R mRNA and protein levels in a time-dependent manner and induced CB(1)R internalization. NF-κB and cPLA(2) were involved in the HG-induced increase in CB(1)R levels. Using a CB(1)R antagonist (AM251) and CB(1) siRNA transfection, we showed that HG-induced CB(1)R is linked to apoptosis. Specifically, HG inhibited the expression of GRP78, but induced increases in endoplasmic reticulum (ER) stress proteins, including phosphorylated (p)-protein kinase-like ER-associated kinase, p-eukaryotic initiation factor 2α, p-activating transcription factor-4, and C/EBP homologous protein. In addition, HG increased the Bax/Bcl-2 ratio and increased the amounts of cleaved poly(ADP-ribose) polymerase and caspase-3. These apoptotic effects were prevented by AM251 and by the downregulation of CB(1)R expression by small interfering RNA. We propose a mechanism by which blockade of CB(1)R attenuates HG-induced apoptosis in rat mesangial cells. Our findings suggest that blockade of CB(1)R may be a potential therapy in diabetic nephropathy.

Effect of protopanaxadiol derivatives in high glucose-induced fibronectin expression in primary cultured rat mesangial cells: role of mitogen-activated protein kinases and Akt.

A lot of anti-diabetic agents using natural plants have been extensively studied. Ginsenosides are known to be used as a remedy for diabetes in Asian countries and American Societies. Diabetic nephropathy is a major complication of diabetes mellitus. Extracellular matrix in mesangial cells is mainly composed of fibronectin and the increase of fibronectin is a hallmark of diabetic nephropathy. Protopenaxadiol (PPD) is a major component of total ginseng. Thus, we examined the regulatory mechanism of PPD derivatives-induced preventive effect of fibronectin expression in mesangial cells cultivated under diabetic condition. In present study, ginsenoside Rb1 prevented the high glucose-induced increase of fibronectin expression in mesangial cells. Ginsenoside Rb2 and Rg3 also mildly inhibited it. However, ginsenoside Rc and Rd did not prevent the high glucose-induced increase of fibronectin expression in mesangial cells. In addition, ginsenoside Rb1 prevented high glucose-induced phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK, JNK/SAPK, and Akt. These results suggest that ginsenoside Rb1 is the most powerful component of PPD derivatives. In conclusion, ginsenoside Rb1 prevented high glucose-induced increase of fibronectin expression via the inhibition of MAPK-Akt signaling cascade.