Upregulation of the ERRγ-VDAC1 axis underlies the molecular pathogenesis of pancreatitis.

Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.

Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This Method.

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.

Tunicamycin as a Novel Redifferentiation Agent in Radioiodine Therapy for Anaplastic Thyroid Cancer.

The silencing of thyroid-related genes presents difficulties in radioiodine therapy for anaplastic thyroid cancers (ATCs). Tunicamycin (TM), an N-linked glycosylation inhibitor, is an anticancer drug. Herein, we investigated TM-induced restoration of responsiveness to radioiodine therapy in radioiodine refractory ATCs. 125I uptake increased in TM-treated ATC cell lines, including BHT101 and CAL62, which was inhibited by KClO4, a sodium-iodide symporter (NIS) inhibitor. TM upregulated the mRNA expression of iodide-handling genes and the protein expression of NIS. TM blocked pERK1/2 phosphorylation in both cell lines, but AKT (protein kinase B) phosphorylation was only observed in CAL62 cells. The downregulation of glucose transporter 1 protein was confirmed in TM-treated cells, with a significant reduction in 18F-fluorodeoxyglucose (FDG) uptake. A significant reduction in colony-forming ability and marked tumor growth inhibition were observed in the combination group. TM was revealed to possess a novel function as a redifferentiation inducer in ATC as it induces the restoration of iodide-handling gene expression and radioiodine avidity, thereby facilitating effective radioiodine therapy.

In Vivo Optical Reporter-Gene-Based Imaging of Macrophage Infiltration of DNCB-Induced Atopic Dermatitis.

This study was conducted to monitor the macrophage infiltration of atopic dermatitis (AD)-like skin lesions and to evaluate the effects of anti-AD therapeutic agents in immunocompetent mice via optical reporter-gene-based molecular imaging. The enhanced firefly luciferase (effluc)-expressing macrophage cell line (Raw264.7/effluc) was intravenously introduced into mice with 2,4-dinitrochlorobenzene (DNCB)-induced AD, followed by bioluminescent imaging (BLI). After in vivo imaging, AD-like skin lesions were excised, and ex vivo imaging and Western blotting were conducted to determine the presence of infused macrophages. Finally, the therapeutic effect of dexamethasone (DEX), an AD-modulating agent, was evaluated via macrophage tracking. In vivo imaging with BLI revealed the migration of the reporter macrophages to DNCB-induced AD-like skin lesions on day 1 post-transfer. The greatest recruitment was observed on day 3, and a decline in BLI signal was observed on day 14. Notably, in vivo BLI clearly showed the inhibition of the reporter macrophage infiltration of DNCB-induced AD-like skin lesions by DEX, which was consistent with the reduced AD symptoms observed in DEX-treated mice. We successfully visualized the macrophage migration to DNCB-induced AD-like skin lesions, proving the feasibility of macrophage imaging for evaluating AD-regulating drugs in living organisms.

Phenylbutyrate Ameliorates High-Fat Diet-Induced Obesity via Brown Adipose Tissue Activation.

Obesity, which is characterized by an excessive accumulation of body fat, is one of the critical factors causing metabolic syndrome. Many studies have been performed to identify appropriate agents to control obesity, but toxicity remains a problem. Herein, we identified that phenylbutyrate (PBA), which has been used to treat urea cycle disorder with very low toxicity for a long time, efficiently inhibited high fat-induced body weight gain in a diet-induced obesity mouse model (DIO model). PBA treatment decreased body fat mass and increased lean composition. Moreover, PBA increased brown adipose tissue (BAT) activity by increasing glucose uptake, thereby improving glucose tolerance and insulin tolerance. Interestingly, PBA could induce the expression of liver type phosphofructokinase (PFKL), a key enzyme in the glycolytic pathway, and knocking down PFKL dramatically repressed the expression level of Ucp1 as well as those of Prdm16, Cidea, Pgc1α, and Pparγ, which are marker genes for BAT activation. These results strongly suggested that PBA could increase energy expenditure by increasing BAT activity via the induction of PFKL. Taken together, PBA could be used as a therapeutic agent for people with obesity to prevent the development of metabolic syndrome.

PEGylated crushed gold shell-radiolabeled core nanoballs for in vivo tumor imaging with dual positron emission tomography and Cerenkov luminescent imaging.

BACKGROUND: Radioactive isotope-labeled gold nanomaterials have potential biomedical applications. Here, we report the synthesis and characterization of PEGylated crushed gold shell-radioactive iodide-124-labeled gold core nanoballs (PEG-124I-Au@AuCBs) for in vivo tumor imaging applications through combined positron emission tomography and Cerenkov luminescent imaging (PET/CLI). RESULTS: PEG-124I-Au@AuCBs showed high stability and sensitivity in various pH solutions, serum, and in vivo conditions and were not toxic to tested cells. Combined PET/CLI clearly revealed tumor lesions at 1 h after injection of particles, and both signals remained visible in tumor lesions at 24 h, consistent with the biodistribution results. CONCLUSION: Taken together, the data provided strong evidence for the application of PEG-124I-Au@AuCBs as promising imaging agents in nuclear medicine imaging of various biological systems, particularly in cancer diagnosis.

Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging.

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(-) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(-) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.

Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) has a high risk of relapse and there are few chemotherapy options. Although 5-hydroxytryptamine (5-HT, serotonin) signaling pathways have been suggested as potential targets for anti-cancer drug development, the mechanism responsible for the action of 5-HT in TNBC remains unknown. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. Cell proliferation was measured using CellTiter 96 Aqueous One Solution. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Levels of 5-HT and vascular endothelial growth factor (VEGF) were measured using ELISA kits. Chick chorioallantoic membrane (CAM) assay and mouse tumor model were used to investigate the in vivo effects of SB269970, a 5-HT7 receptor antagonist, and BJ-1113, a novel synthetic compound. RESULTS: TNBC cell lines (MDA-MB-231, HCC-1395, and Hs578T) expressed higher levels of tryptophan hydroxylase 1 (TPH1) than hormone-responsive breast cancer cell lines (MCF-7 and T47D). In MDA-MB-231 cells, 5-HT promoted invasion and proliferation via 5-HT7 receptor, and interestingly, the stimulatory effect of 5-HT on MDA-MB-231 cell invasion was stronger than its effect on proliferation. Likewise, downstream signaling pathways of 5-HT7 differed during invasion and proliferation, that is, Gα-activated cAMP and Gßγ-activated kinase signaling during invasion, and Gßγ-activated PI3K/Akt signaling during proliferation. Also, 5-HT increased the protein expressions of TPH1 and VEGF in MDA-MB-231 cells. These results provide insight of the stimulatory effect of 5-HT on breast cancer progression; 5-HT was found to act more strongly during the first stage of metastasis (during invasion and migration) than during the later proliferative phase after local invasion. Interestingly, these actions of 5-HT were inhibited by BJ-1113, a 6-amino-2,4,5-trimethylpyridin-3-ol analog. BJ-1113 blocked intracellular signaling pathways initiated by 5-HT7 receptor activation, and exhibited anti-proliferative and anti-invasive activities against MDA-MB-231 cells. Furthermore, the inhibitory effect of BJ-1113 against MDA-MB-231 tumor growth was greater than that of SB269970, a 5-HT7 receptor antagonist. CONCLUSIONS: 5-HT7 receptor which mediates 5-HT-induced cancer progression is a potential therapeutic target in TNBC, and BJ-1113 offers a novel scaffold for the development of anti-cancer agents against TNBC.

Combined Positron Emission Tomography and Cerenkov Luminescence Imaging of Sentinel Lymph Nodes Using PEGylated Radionuclide-Embedded Gold Nanoparticles.

New imaging probes with high sensitivity and stability are urgently needed to accurately detect sentinel lymph nodes (SLNs) for successful cancer diagnosis. Herein, the use of highly sensitive and stable PEGylated radionuclide-embedded gold nanoparticles (PEG-RIe-AuNPs) is reported for the detection of SLNs by combined positron emission tomography and Cerenkov luminescence imaging (PET/CLI). PEG-RIe-AuNPs show high sensitivity and stability both in vitro and in vivo, and are not toxic to normal ovarian and immune cells. In vivo PET/CLI imaging clearly reveals SLNs as early as 1 h post PEG-RIe-AuNP-injection, with peak signals achieved at 6 h postinjection, which is consistent with the biodistribution results. Taken together, the data provide strong evidence that PEG-RIe-AuNPs are promising as potential lymphatic tracers in biomedical imaging for pre and intraoperative surgical guidance.

Retinoic acid-related orphan receptor alpha reprograms glucose metabolism in glutamine-deficient hepatoma cells.

UNLABELLED: The metabolism of glutamine and glucose is recognized as a promising therapeutic target for the treatment of cancer; however, targeted molecules that mediate glutamine and glucose metabolism in cancer cells have not been addressed. Here, we show that restricting the supply of glutamine in hepatoma cells, including HepG2 and Hep3B cells, markedly increased the expression of retinoic acid-related orphan receptor alpha (RORα). Up-regulation of RORα in glutamine-deficient hepatoma cells resulted from an increase in the level of cellular reactive oxygen species and in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate reduced (NADP+ /NADPH) ratio, which was consistent with a reduction in the glutathione/glutathione disulfide (GSH/GSSG) ratio. Adenovirus (Ad)-mediated overexpression of RORα (Ad-RORα) or treatment with the RORα activator, SR1078, reduced aerobic glycolysis and down-regulated biosynthetic pathways in hepatoma cells. Ad-RORα and SR1078 reduced the expression of pyruvate dehydrogenase kinase 2 (PDK2) and inhibited the phosphorylation of pyruvate dehydrogenase and subsequently shifted pyruvate to complete oxidation. The RORα-mediated decrease in PDK2 levels was caused by up-regulation of p21, rather than p53. Furthermore, RORα inhibited hepatoma growth both in vitro and in a xenograft model in vivo. We also found that suppression of PDK2 inhibited hepatoma growth in a xenograft model. These findings mimic the altered glucose utilization and hepatoma growth caused by glutamine deprivation. Finally, tumor tissue from 187 hepatocellular carcinoma patients expressed lower levels of RORα than adjacent nontumor tissue, supporting a potential beneficial effect of RORα activation in the treatment of liver cancer. CONCLUSION: RORα mediates reprogramming of glucose metabolism in hepatoma cells in response to glutamine deficiency. The relationships established here between glutamine metabolism, RORα expression and signaling, and aerobic glycolysis have implications for therapeutic targeting of liver cancer metabolism.

Identification of Selective ERRγ Inverse Agonists.

GSK5182 (4) is currently one of the lead compounds for the development of estrogen-related receptor gamma (ERRγ) inverse agonists. Here, we report the design, synthesis, pharmacological and in vitro absorption, distribution, metabolism, excretion, toxicity (ADMET) properties of a series of compounds related to 4. Starting from 4, a series of analogs were structurally modified and their ERRγ inverse agonist activity was measured. A key pharmacophore feature of this novel class of ligands is the introduction of a heterocyclic group for A-ring substitution in the core scaffold. Among the tested compounds, several of them are potent ERRγ inverse agonists as determined by binding and functional assays. The most promising compound, 15g, had excellent binding selectivity over related subtypes (IC50 = 0.44, >10, >10, and 10 µM at the ERRγ, ERRα, ERRß, and ERα subtypes, respectively). Compound 15g also resulted in 95% transcriptional repression at a concentration of 10 µM, while still maintaining an acceptable in vitro ADMET profile. This novel class of ERRγ inverse agonists shows promise in the development of drugs targeting ERRγ-related diseases.

Anatomical configuration of the inferior alveolar neurovascular bundle: a histomorphometric analysis.

PURPOSE: The inferior alveolar neurovascular bundle (NVB) is important in implant placement and many other surgeries in dentistry because it is a major supplier of sensation and blood to the mandible via the mandibular canal. The purposes of the present study were to determine the areas and diameters of the NVB, the inferior alveolar nerve (IAN), and the inferior alveolar artery (IAA), and to verify the buccolingual location of the mandibular canal. METHODS: The anatomical configuration of the NVB was examined by histomorphometrically analyzing 20 embalmed dentulous hemimandibles. The areas and maximum horizontal and vertical diameters of the NVB, IAN, and IAA were measured according to tooth region. The distances from the internal border of the mandibular canal to the outer surface of the buccal and lingual cortical plates were also measured. RESULTS: The areas of the vertically oval-shaped NVB and IAN appeared to be constant between the molar and premolar regions, which contain the mental branch, and decreased sharply in the lateral incisor after branching off of the mental branch via the mental canal. The mandibular canal was located close to the lingual cortical plate in the posterior tooth region before passing through the mental canal, immediately after which it was situated quite close to the buccal cortical plate, and then closer to the middle toward the anterior tooth region. CONCLUSIONS: The findings of this study provide useful anatomical information that should help to minimize the risk of injury to the NVB during surgical procedures in the mandibular region.

Suppression of tumorigenesis in mitochondrial NADP(+)-dependent isocitrate dehydrogenase knock-out mice.

The tumor host microenvironment is increasingly viewed as an important contributor to tumor growth and suppression. Cellular oxidative stress resulting from high levels of reactive oxygen species (ROS) contributes to various processes involved in the development and progress of malignant tumors including carcinogenesis, aberrant growth, metastasis, and angiogenesis. In this regard, the stroma induces oxidative stress in adjacent tumor cells, and this in turn causes several changes in tumor cells including modulation of the redox status, inhibition of cell proliferation, and induction of apoptotic or necrotic cell death. Because the levels of ROS are determined by a balance between ROS generation and ROS detoxification, disruption of this system will result in increased or decreased ROS level. Recently, we demonstrated that the control of mitochondrial redox balance and cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) that supplies NADPH for antioxidant systems. To explore the interactions between tumor cells and the host, we evaluated tumorigenesis between IDH2-deficient (knock-out) and wild-type mice in which B16F10 melanoma cells had been implanted. Suppression of B16F10 cell tumorigenesis was reproducibly observed in the IDH2-deficient mice along with significant elevation of oxidative stress in both the tumor and the stroma. In addition, the expression of angiogenesis markers was significantly down-regulated in both the tumor and the stroma of the IDH2-deficient mice. These results support the hypothesis that redox status-associated changes in the host environment of tumor-bearing mice may contribute to cancer progression.

Brazilin Isolated from Caesalpinia sappan suppresses nuclear envelope reassembly by inhibiting barrier-to-autointegration factor phosphorylation.

To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.

Topographical relationship of the greater palatine artery and the palatal spine. Significance for periodontal surgery.

AIM: The aims of this study were to (1) identify the branching pattern and course of the greater palatine artery (GPA), (2) carry out a morphological analysis of the palatal bony prominence that divides the medial and lateral grooves and (3) characterize the topographical relationships between these two structures. METHODS: Thirty-six hemimaxillae were studied with the aid of a surgical microscope to elucidate the GPA. A further 25 dry skulls were examined to establish the morphology of the palatal spine. RESULTS: The most common GPA branching pattern was type I (41.7%, 15 sides), which gave off the medial and canine branches after the bony prominence. The distances from the CEJ to the lateral branch of the GPA were 9.04 ± 2.93 mm (canine), 11.12 ± 1.89 mm (first premolar), 13.51 ± 2.08 mm (second premolar), 13.76 ± 2.86 mm (first molar) and 13.91 ± 2.20 mm (second molar). The palatal spine was frequently observed as the bony prominence (66.3%, 57 sides), and was located at 6.49 ± 1.76 mm from the greater palatine foramen, with a length of 10.42 ± 2.45 mm. There was no a correlation between the bony prominence shape and the GPA branching pattern. CONCLUSIONS: These results could provide the reference data regarding the topography of the GPA for periodontal surgery.

Remodeling of sorafenib as an orally bioavailable ferroptosis inducer for Lung Cancer by chemical modification of adenine-binding motif.

Sorafenib (BAY 43-9006) was developed as a multi-kinase inhibitor to treat advanced renal cell, hepatocellular, and thyroid cancers. The cytotoxic effect of sorafenib on cancer cells results from not only inhibiting the MEK/ERK signaling pathway (the on-target effect) but also inducing oxidative damage (the off-target effect). The inhibitory effect of sorafenib on system Xc- (xCT), a cystine/glutamate antiporter, promotes ferroptosis induction and accounts for oxidative damage. While emerging studies on ferroptosis in cancers have garnered increasing attention, the lack of consideration for ferroptosis inducers (FINs) with favorable pharmacokinetics could be problematic. Herein, we remodeled the chemical structure of sorafenib, of which pharmacokinetics and safety are already assured, to customize the off-target effect (i.e., ferroptosis induction) to on-target by disrupting the adenine-binding motif. JB3, a sorafenib derivative (i.e., JB compounds), with a tenfold higher IC50 toward RAF1 because of chemical remodeling, induced strong cytotoxicity in the elastin-sensitive lung cancer cells, while it was markedly reduced by ferrostatin-1. The 24% oral bioavailability of JB3 in rats accounted for a significant anti-tumor effect of orally administrated JB3 in xenograft models. These results indicate that JB3 could be further developed as an orally bioavailable FIN in novel anti-cancer therapeutics.

The combination of ANT2 shRNA and hNIS radioiodine gene therapy increases CTL cytotoxic activity through the phenotypic modulation of cancer cells: combination treatment with ANT2 shRNA and I-131.

BACKGROUND: It is important to simultaneously induce strong cell death and antitumor immunity in cancer patients for successful cancer treatment. Here, we investigated the cytotoxic and phenotypic modulation effects of the combination of ANT2 shRNA and human sodium iodide symporter (hNIS) radioiodine gene therapy in vitro and in vivo and visualized the antitumor effects in an immunocompromised mouse colon cancer model. METHODS: A mouse colon cancer cell line co-expressing hNIS and the luciferase gene (CT26/hNIS-Fluc, named CT26/NF) was established. CT26/NF cells and tumor-bearing mice were treated with HBSS, scramble, ANT2 shRNA, I-131, and ANT2 shRNA + I-131. The apoptotic rates (%) and MHC class I and Fas gene expression levels were determined in treated CT26/NF cells using flow cytometry. Concurrently, the level of caspase-3 activation was determined in treated cells in vitro. For in vivo therapy, tumor-bearing mice were treated with scramble, ANT2 shRNA, I-131, and the combination therapy, and the anti-tumor effects were monitored using bioluminescence. The killing activity of cytotoxic T cells (CTLs) was measured with a lactate dehydrogenase (LDH) assay. RESULTS: For the in vitro experiments, the combination of ANT2 shRNA and I-131 resulted in a higher apoptotic cell death rate compared with ANT2 shRNA or I-131 alone, and the levels of MHC class I and Fas-expressing cancer cells were highest in the cells receiving combination treatment, while single treatment modestly increased the level of MHC class I and Fas gene expression. The combination of ANT2 shRNA and I-131 resulted in a higher caspase-3 activation than single treatments. Interestingly, in vivo combination treatment led to increased gene expression of MHC class I and Fas than the respective mono-therapies; furthermore, bioluminescence showed increased antitumor effects after combination treatment than monotherapies. The LDH assay revealed that the CTL killing activity against CT26/NF cells was most effective after combination therapy. CONCLUSIONS: Increased cell death and phenotypic modulation of cancer cells in vitro and in vivo were achieved simultaneously after combination therapy with ANT2 shRNA and I-131, and this combination therapy induced remarkable antitumor outcomes through improvements in CTL immunity against CT26/NF. Our results suggest that combination therapy can be used as a new therapeutic strategy for cancer patients who show resistance to single therapy such as radiation or immunotherapy.

An Inverse Agonist of Estrogen-Related Receptor Gamma, GSK5182, Enhances Na+/I- Symporter Function in Radioiodine-Refractory Papillary Thyroid Cancer Cells.

Previously, we reported that an inverse agonist of estrogen-related receptor gamma (ERRγ), GSK5182, enhances sodium iodide (Na+/I-) symporter (NIS) function through mitogen-activated protein (MAP) kinase signaling in anaplastic thyroid cancer cells. This finding helped us to further investigate the effects of GSK5182 on NIS function in papillary thyroid cancer (PTC) refractory to radioactive iodine (RAI) therapy. Herein, we report the effects of ERRγ on the regulation of NIS function in RAI-resistant PTC cells using GSK5182. RAI-refractory BCPAP cells were treated with GK5182 for 24 h at various concentrations, and radioiodine avidity was determined with or without potassium perchlorate (KClO4) as an NIS inhibitor. We explored the effects of GSK5182 on ERRγ, the mitogen-activated protein (MAP) kinase pathway, and iodide metabolism-related genes. We examined whether the MAP pathway affected GSK5182-mediated NIS function using U0126, a selective MEK inhibitor. A clonogenic assay was performed to evaluate the cytotoxic effects of I-131. GSK5182 induced an increase in radioiodine avidity in a dose-dependent manner, and the enhanced uptake was completely inhibited by KClO4 in BCPAP cells. We found that ERRγ was downregulated and phosphorylated extracellular signal-regulated kinase (ERK)1/2 was upregulated in BCPAP cells, with an increase in total and membranous NIS and iodide metabolism-related genes. MEK inhibitors reversed the increase in radioiodine avidity induced by GSK5182. Clonogenic examination revealed the lowest survival in cells treated with a combination of GSK5182 and I-131 compared to those treated with either GSK518 or I-131 alone. We demonstrate that an inverse agonist of ERRγ, GSK5182, enhances the function of NIS protein via the modulation of ERRγ and MAP kinase signaling, thereby leading to increased responsiveness to radioiodine in RAI-refractory papillary thyroid cancer cells.

Visualizing mast cell migration to tumor sites using sodium iodide symporter of nuclear medicine reporter gene.

PURPOSE: Owing to the close relationship between mast cells and cancer progression, an imaging technique that can be applied in a clinical setting to explore the biological behavior of mast cells in the tumor microenvironment is needed. In this study, we visualized mast cell migration to lung tumor lesions in live mice using sodium iodide symporter (NIS) as a nuclear medicine reporter gene. EXPERIMENTAL DESIGN: The murine mast cell line MC-9 was infected with retrovirus including NIS, luciferase (as a surrogate marker for NIS), and Thy1.1 to generate MC-9/NFT cells. Radioiodine uptake was measured in MC-9/NFT cells, and an inhibition assay of radioiodine uptake using KCLO4 was also performed. Cell proliferation and FcεRI expression was examined in MC-9 and MC-9/NFT cells. The effect of mast cell-conditioned media (CM) on the proliferation of Lewis lung cancer (LLC) cells was examined. The migration level of MC-9/NFT cells was confirmed in the presence of serum-free media (SFM) and CM of cancer cells. After intravenous injection of MC-9/NFT cells into mice with an LLC tumor, I-124 PET/CT and biodistribution analysis was performed. RESULTS: MC-9/NFT cells exhibited higher radioiodine avidity compared to parental MC-9 cells; this increased radioiodine avidity in MC-9/NFT cells was reduced to basal level by KCLO4. Levels of FcεRI expression and cell proliferation were not different in parental MC-9 cell and MC-9/ NFT cells. The CM of MC-9/NFT cells increased cancer cell proliferation relative to that of the SFM. The migration level of MC-9/NFT cells was higher in the CM than the SFM of LLC cells. PET/CT imaging with I-124 clearly showed infiltration of reporter mast cells in lung tumor at 24 h after transfer, which was consistent with the findings of the biodistribution examination. CONCLUSION: These findings suggest that the sodium iodide symporter can serve as a reliable nuclear medicine reporter gene for non-invasively imaging the biological activity of mast cells in mice with lung tumors. Visualizing mast cells in the tumor microenvironment via a nuclear medicine reporter gene would provide valuable insights into their biological functions.

Discovery and Feasibility Study of Medical Fluorophore 33 as a Novel Theranostic Agent.

Fluorescent dyes have garnered significant attention as theranostic platforms owing to their inherent characteristics. In this study, we present the discovery of Medical Fluorophore 33 (MF33), a novel and potent theranostic agent with a phenaleno-isoquinolinium salt structure that can serve as a cancer therapeutic strategy. The synthesis of MF33 is readily achievable through a simple Rh(III)-catalyzed reaction. Moreover, MF33 displayed strong fluorescence signals, excellent microsomal stability, and high biocompatibility in vivo. It induces significant apoptosis in cancer cells via the p53/p21/caspase-3 signaling pathway, leading to selective cytotoxicity in various cancer cells. In vivo fluorescence imaging with MF33 enabled the visualization of sentinel lymph nodes in living mice. Notably, repeated intraperitoneal administration of MF33 resulted in antitumor activity in mice with colorectal cancer. Collectively, our findings suggest that phenaleno-isoquinolinium salt-based MF33 is a viable theranostic agent for biomedical imaging and cancer treatment.