Decoding the temporal nature of brain GR activity in the NFκB signal transition leading to depressive-like behavior.

The fine-tuning of neuroinflammation is crucial for brain homeostasis as well as its immune response. The transcription factor, nuclear factor-κ-B (NFκB) is a key inflammatory player that is antagonized via anti-inflammatory actions exerted by the glucocorticoid receptor (GR). However, technical limitations have restricted our understanding of how GR is involved in the dynamics of NFκB in vivo. In this study, we used an improved lentiviral-based reporter to elucidate the time course of NFκB and GR activities during behavioral changes from sickness to depression induced by a systemic lipopolysaccharide challenge. The trajectory of NFκB activity established a behavioral basis for the NFκB signal transition involved in three phases, sickness-early-phase, normal-middle-phase, and depressive-like-late-phase. The temporal shift in brain GR activity was differentially involved in the transition of NFκB signals during the normal and depressive-like phases. The middle-phase GR effectively inhibited NFκB in a glucocorticoid-dependent manner, but the late-phase GR had no inhibitory action. Furthermore, we revealed the cryptic role of basal GR activity in the early NFκB signal transition, as evidenced by the fact that blocking GR activity with RU486 led to early depressive-like episodes through the emergence of the brain NFκB activity. These results highlight the inhibitory action of GR on NFκB by the basal and activated hypothalamic-pituitary-adrenal (HPA)-axis during body-to-brain inflammatory spread, providing clues about molecular mechanisms underlying systemic inflammation caused by such as COVID-19 infection, leading to depression.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Antidepressant-Like Effects of Lindera obtusiloba Extracts on the Immobility Behavior of Rats in the Forced Swim Test.

Lindera obtusiloba extracts are commonly used as an alternative medicine due to its numerous health benefits in Korea. However, the antidepressant-like effects of L. obtusiloba extracts have not been fully elucidated. In this study, we aimed to determine whether L. obtusiloba extracts exhibited antidepressant-like activity in rats subjected to forced swim test (FST)-induced depression. Acute treatment of rats with L. obtusiloba extracts (200 mg/kg, p.o.) significantly reduced immobility time and increased swimming time without any significant change in climbing. Rats treated with L. obtusiloba extracts also exhibited a decrease in the limbic hypothalamic-pituitary-adrenal (HPA) axis response to the FST, as indicated by attenuation of the corticosterone response and decreased c-Fos immunoreactivity in the hippocampus CA3 region. In addition, L. obtusiloba extracts, at concentrations that were not affected by cell viability, significantly decreased luciferase activity in response to cortisol in a concentration-dependent manner by the glucocorticoid binding assay in HeLa cells. Our findings suggested that the antidepressant-like effects of L. obtusiloba extracts were likely mediated via the glucocorticoid receptor (GR). Further studies are needed to evaluate the potential of L. obtusiloba extracts as an alternative therapeutic approach for the treatment of depression.

ß-Caryophyllene potently inhibits solid tumor growth and lymph node metastasis of B16F10 melanoma cells in high-fat diet-induced obese C57BL/6N mice.

We reported previously that high-fat diet (HFD) feeding stimulated solid tumor growth and lymph node (LN) metastasis in C57BL/6N mice injected with B16F10 melanoma cells. ß-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in many essential oils and has been shown to exert anti-inflammatory activities. To examine whether BCP inhibits HFD-induced melanoma progression, 4-weeks old, male C57BL/6N mice were fed a control diet (CD, 10 kcal% fat) or HFD (60 kcal% fat + 0, 0.15 or 0.3% BCP) for the entire experimental period. After 16 weeks of feeding, B16F10s were subcutaneously injected into mice. Three weeks later, tumors were resected, and mice were killed 2 weeks post-resection. Although HFD feeding increased body weight gain, fasting blood glucose levels, solid tumor growth, LN metastasis, tumor cell proliferation, angiogenesis and lymphangiogenesis, it decreased apoptotic cells, all of which were suppressed by dietary BCP. HFD feeding increased the number of lipid vacuoles and F4/80+ macrophage (MΦ) and macrophage mannose receptor (MMR)+ M2-MΦs in tumor tissues and adipose tissues surrounding the LN, which was suppressed by BCP. HFD feeding increased the levels of CCL19 and CCL21 in the LN and the expression of CCR7 in the tumor; these changes were blocked by dietary BCP. In vitro culture results revealed that BCP inhibited lipid accumulation in 3T3-L1 preadipocytes; monocyte migration and monocyte chemoattractant protein-1 secretion by B16F10s, adipocytes and M2-MΦs; angiogenesis and lymphangiogenesis. The suppression of adipocyte and M2-cell accumulation and the inhibition of CCL19/21-CCR7 axis may be a part of mechanisms for the BCP suppression of HFD-stimulated melanoma progression.

High-fat diet-induced obesity increases lymphangiogenesis and lymph node metastasis in the B16F10 melanoma allograft model: roles of adipocytes and M2-macrophages.

To examine the effects of high-fat diet (HFD) on melanoma progression, HFD-fed C57BL/6N mice were subcutaneously injected with syngeneic B16F10 melanoma cells. At 3 weeks post-injection, the tumors were resected; the mice were then sacrificed at 2 weeks post-resection. HFD stimulated melanoma growth and lymph node (LN) metastasis as well as tumor and LN lymphangiogenesis. Lipid vacuoles in the tumor and M2-macrophage (MΦ)s in the adipose and tumor tissues were increased in HFD-fed mice. CCL19 and CCL21 contents were higher in LNs than in tumors. HFD increased both CCL19 and CCL21 levels in LNs and CCR7 in tumors. Adipose tissue-conditioned media (CM) from HFD-fed mice enhanced lymphangiogenesis, and mature adipocyte (MA)/M2-MΦ co-culture CM markedly stimulated the tube formation of lymphatic endothelial cell (LEC)s and B16F10 migration. Monocyte migration was moderately stimulated by B16F10 or MA CM, but tremendously stimulated by B16F10/M2-MΦ co-culture CM, which was enhanced by MA/B16F10/M2-MΦ co-culture CM. The co-culture results revealed that MAs increased CCL2, M-CSF and CCR7 mRNAs in B16F10s; vascular endothelial growth factor (VEGF)-D mRNA in M2-MΦs; and CCL19, CCL21 and VEGF receptor (VEGFR)3 mRNA in LECs. M2-MΦs increased CCL2, M-CSF and VEGF-A mRNAs in B16F10s, whereas B16F10s increased VEGF-C mRNAs in M2-MΦs and VEGFR3 mRNA in LECs. These results indicate that in HFD-fed mice, MA-induced CCL2 and M-CSF in tumor cells increase M2-MΦs in tumor; the crosstalk between tumor cells and M2-MΦs further increases cytokines and angiogenic and lymphangiogenic factors. Additionally, MA-stimulated CCL19, CCL21/CCR7 axis contributes to increased LN metastasis in HFD-fed mice.

Benzyl isothiocyanate suppresses high-fat diet-stimulated mammary tumor progression via the alteration of tumor microenvironments in obesity-resistant BALB/c mice.

We previously reported that a high-fat diet (HFD) and M2-macrophages induce changes in tumor microenvironments and stimulate tumor growth and metastasis of 4T1 mammary cancer cells in BALB/c mice. In this study, we attempted to determine whether benzyl isothiocyanate (BITC) inhibits HFD-induced changes in tumor progression and in tumor microenvironments. Four groups of female BALB/c mice (4-week-old) were fed on a control diet (CD, 10 kcal% fat) and HFD (60 kcal% fat) containing BITC (0, 25, or 100 mg/kg diet) for 20 weeks. Following 16 weeks of feeding, 4T1 cells (5×10(4) cells) were injected into the mammary fat pads, and animals were killed 30 d after the injection. HFD feeding increased solid tumor growth and the number of tumor nodules in the lung and liver, as compared to the CD group, and these increases were inhibited by BITC supplementation. The number of lipid vacuoles, CD45+ leukocytes and CD206+ M2-macrophages, expression of Ki67, levels of cytokines/chemokines, including macrophage-colony stimulating factor (M-CSF) and monocyte chemoattractant protein-1, and mRNA levels of F4/80, CD86, Ym1, CD163, CCR2, and M-CSF receptor were increased in the tumor tissues of HFD-fed mice, and these increases were inhibited by BITC supplementation. In vitro culture results demonstrated that BITC inhibited macrophage migration as well as lipid droplet accumulation in 3T3-L1 cells. These results suggest that suppression of lipid accumulation and macrophage infiltration in tumor tissues may be one of the mechanisms by which BITC suppresses tumor progression in HFD-fed mice.

Neuroprotection of posttreatment with risperidone, an atypical antipsychotic drug, in rat and gerbil models of ischemic stroke and the maintenance of antioxidants in a gerbil model of ischemic stroke.

Risperidone, an atypical antipsychotic drug, has been discovered to have some beneficial effects beyond its original effectiveness. The present study examines the neuroprotective effects of risperidone against ischemic damage in the rat and gerbil induced by transient focal and global cerebral ischemia, respectively. The results showed that pre- and posttreatment with 4 mg/kg risperidone significantly protected against neuronal death from ischemic injury. Many NeuN-immunoreactive neurons and a few F-J B-positive cells were found in the rat cerebral cortex and gerbil hippocampal CA1 region (CA1) in the risperidone-treated ischemia groups compared with those in the vehicle-treated ischemia group. In addition, treatment with risperidone markedly attenuated the activation of microglia in the gerbil CA1. On the other hand, we found that treatment with risperidone significantly maintained the antioxidants levels in the ischemic gerbil CA1. Immunoreactivities of superoxide dismutases 1 and 2, catalase, and glutathione peroxidase were maintained in the stratum pyramidale of the CA1; the antioxidants were very different from those in the vehicle-treated ischemia groups. In brief, our present findings indicate that posttreatment as well as pretreatment with risperidone can protect neurons in the rat cerebral cortex and gerbils CA1 from transient cerebral ischemic injury and that the neuroprotective effect of risperidone may be related to attenuation of microglial activation as well as maintenance of antioxidants.

Polyamines cause elevation of steroid 5α-reductase mRNA levels by suppressing mRNA degradation in C6 glioma cells.

Polyamines are widely distributed in living organisms, and considered to play a potential role in various cellular processes. The effects of polyamines on gene expression as well as cell proliferation have been suggested to be closely associated with the physiological and pathological functions. However, it seems necessary to investigate their potential roles in the regulation of cellular metabolism and functions. Previously, glial cells have been suggested to be involved in the protection and preservation of neuronal functions, probably through the production of neurotrophic factors in the brain. On the other hand, neuroactive 5α-reduced steroids promote glial cell differentiation, resulting in enhancement of their ability to produce brain-derived neurotrophic factor (BDNF). Based on these findings, polyamines are assumed to stimulate the expression of the gene encoding steroid 5α-reductase (5α-R), which can induce the production of neuroactive 5α-reduced steroids in glial cells. The effects of polyamines on 5α-R mRNA levels in C6 glioma cells were examined as a model experiment. In consequence, spermine (SPM) and spermidine (SPD), but not putrescine (PUT), have been shown to elevate 5α-R mRNA levels without activating the 5α-R promoter. Furthermore, SPM increased 5α-R mRNA levels under the conditions in which the mRNA biosynthesis was inhibited. Therefore, it can be speculated that polyamines increase 5α-R mRNA levels as a consequence of suppressing the degradation of mRNA.

Palytoxin causes nonoxidative necrotic damage to PC12 cells in culture.

Palytoxin (PTX) is a potent marine toxin that causies serious damage to various tissues and organs. It has been reported to affect the transport of cations across the plasma membranes, which is commonly recognized as being the principal mechanism of its highly toxic action on mammals, including humans. However, although some marine toxins have been shown to cause toxic effects on the nervous system by interfering with the transmission of nerve impulses, the effect of PTX on neuronal cells has not yet been fully elucidated. Therefore, the toxic action of PTX on PC12 cells was examined as an in vitro model experiment to elucidate the neurotoxic properties of this toxin, and PTX was shown to reduce the viability of PC12 cells in a concentration-dependent manner. The cytotoxic action of PTX was not significantly altered by the presence of the antioxidant N-acetylcysteine and reduced-form glutathione in the cultures. Fluorescence staining of the cells and the electrophoretic analysis of genomic DNA showed that PTX failed to cause chromatin condensation and DNA fragmentation within the cells. On the other hand, the exposure to PTX caused positive staining of the cytoplasmic space of the cells with propidium iodide and the release of lactate dehydrogenase into the culture medium. Based on these observations, PTX is considered to cause cell death as a consequence of disrupting the plasma membranes, thus causing nonoxidative necrotic damage to PC12 cells.

Stress coping stimulates hippocampal neurogenesis in adult monkeys.

Coping with intermittent social stress is an essential aspect of living in complex social environments. Coping tends to counteract the deleterious effects of stress and is thought to induce neuroadaptations in corticolimbic brain systems. Here we test this hypothesis in adult squirrel monkey males exposed to intermittent social separations and new pair formations. These manipulations simulate conditions that typically occur in male social associations because of competition for limited access to residency in mixed-sex groups. As evidence of coping, we previously confirmed that cortisol levels initially increase and then are restored to prestress levels within several days of each separation and new pair formation. Follow-up studies with exogenous cortisol further established that feedback regulation of the hypothalamic-pituitary-adrenal axis is not impaired. Now we report that exposure to intermittent social separations and new pair formations increased hippocampal neurogenesis in squirrel monkey males. Hippocampal neurogenesis in rodents contributes to spatial learning performance, and in monkeys we found that spatial learning was enhanced in conditions that increased hippocampal neurogenesis. Corresponding changes were discerned in the expression of genes involved in survival and integration of adult-born granule cells into hippocampal neural circuits. These findings support recent indications that stress coping stimulates hippocampal neurogenesis in adult rodents. Psychotherapies designed to promote stress coping potentially have similar effects in humans with major depression.

Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model.

INTRODUCTION: Tumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors. METHODS: 4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted. RESULTS: The co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone. CONCLUSION: The crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo.

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.

The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction.

Autophagy is a membrane trafficking process involved in intracellular degradation and recycling in eukaryotic cells. DRAM2 (damage-regulated autophagy modulator 2) is a homologue of DRAM that regulates p53-mediated cell death. As its name implies, DRAM expression induces autophagy in a p53-dependent manner; however, the role of DRAM2 in autophagy is not clear. In this study, we report that DRAM2 expression contributes to autophagy induction. Overexpression of DRAM2 induces cytoplasmic GFP-LC3 punctuates, and increases the level of endogenous LC3-II. Moreover, the silencing of endogenous DRAM2 interferes with starvation-induced autophagy. Thus, we propose that DRAM2 as well as DRAM are involved in autophagy.

Intracellular ATP assay of live cells using PTD-conjugated luciferase.

Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10(-9) M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays.

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia.

Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damages in the brain. In the present study, ischemia-induced changes in GLP-1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP-1R agonist exendin-4 (EX-4) against ischemic damage was studied. GLP-1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP-1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP-1R protein level was increased again at 10 days after I/R, and GLP-1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX-4 treatment attenuated ischemia-induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose-dependent manner. EX-4 treatment also induced the elevation of GLP-1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP-1R is altered in the ischemic region after an ischemic insult and that EX-4 protects against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglial activation against transient cerebral ischemic damage.

Time-course alterations of Toll-like receptor 4 and NF-κB p65, and their co-expression in the gerbil hippocampal CA1 region after transient cerebral ischemia.

Innate immune system is very important to modulate the host defense against a large variety of pathogens. Toll-like receptors (TLRs) play a key role in controlling innate immune response. Among TLRs, TLR4 is a specific receptor for lipopolysaccharide and associated with the release of pro-inflammatory cytokines. In the present study, we investigated ischemia-related changes of TLR4 immunoreactivity and its protein level, and nuclear factor κB (NF-κB) p65 immunoreactivity regarding inflammatory responses in the hippocampal CA1 region after 5 min of transient cerebral ischemia to identify the correlation between transient ischemia and inflammation. In the sham-operated group, TLR4 immunoreactivity was easily detected in pyramidal neurons of the hippocampal CA1 region (CA1). TLR4 immunoreactivity in pyramidal neurons was distinctively decreased after ischemia/reperfusion (I/R); instead, based on double immunofluorescence study, TLR4 immunoreactivity was expressed in non-pyramidal neurons and astrocytes from 2 days postischemia. In addition, TLR4 protein level was lowest at 1 day postischemia and highest 4 days after I/R. On the other hand, NF-κB p65 immunoreactivity was not detected in the CA1 of the sham-operated group, and NF-κB p65 immunoreactivity was not observed until 1 day after I/R. However, NF-κB p65 immunoreactivity began to be expressed in astrocytes at 2 days postischemia, and the immunoreactivity was strong 4 days postischemia. Our results indicate that TLR4 and NF-κB p65 immunoreactivity are changed in CA1 pyramidal neurons and newly expressed in astrocytes, not in microglia, in the CA1 region after transient cerebral ischemia.

Pharmacokinetics and biodistribution of human serum albumin-TIMP-2 fusion protein using near-infrared optical imaging.

PURPOSE: TIMP-2 has been studied as an attractive cancer therapeutic candidate, and a TIMP-2 fusion protein (HSA/TIMP-2) displayed effective anticancer activity, despite a lack of information about its pharmacokinetics (PK) and biodistribution. The purpose of this work was to assess the PK and biodistribution of HSA/TIMP-2 as well as to quantify accumulated HSA/TIMP-2 in tumors. METHODS: Cy5.5 near-infrared (NIR) fluorescence was conjugated to the HSA/TIMP-2 protein (Cy5.5-HSA/TIMP-2) for monitoring spatio-temporal changes in vivo. For PK and biodistribution analysis, 0.2 µg/g body weight of Cy5.5-HSA/TIMP-2 was injected into MAT-LyLu prostate tumor xenografts, which were then imaged using an IVIS-200 optical imaging system. To quantify the accumulated HSA/TIMP-2 in tumors, we introduced a standard curve with depth-corrected fluorescence measurement. RESULTS: In the vascular tube formation assay with human umbilical vein endothelial cells (HUVECs), Cy5.5-HSA/TIMP-2 showed an antiangiogenic effect. In prostate cancer xenografts, Cy5.5-HSA/TIMP-2 exhibited a prolongation of blood half-life to 19.6 h and relatively preferential distribution to the tumor. The amount of tumor-accumulated Cy5.5-HSA/TIMP-2 was calculated to be 4.5 ± 0.5 ng/g body weight at 2 days, representing 2.25 ± 0.25% of the initial dose. CONCLUSIONS: We evaluated the pharmacokinetic profile and biodistribution of HSA/TIMP-2 with favorable results, providing new information for more effective approaches to cancer therapeutics using HSA/TIMP-2. Additionally, real-time in vivo fluorescence imaging analysis using a depth-corrected standard curve may serve as a platform to quantify biodistributed drug in anticancer therapeutic studies.

Quantification of cellular uptake and in vivo tracking of transduction using real-time monitoring.

Protein transduction domains (PTDs) are short amino acid sequences that promote their own translocation across the cell plasma membrane and have been studied for possible use in drug delivery and gene therapy. However, no direct method to quantify transduction is available. Here, using a new luciferase-tagged human PTD, we show that cellular uptake levels can be determined in a reliable manner. Furthermore, we show that enhanced in vivo tracking by human PTD can be quantified in a mouse model. This is the first report on the direct quantification of PTD transduction in vitro and in vivo, which will be necessary for studying its possible therapeutic application in drug delivery and gene therapy.

Caffeic acid phenethyl ester, a component of beehive propolis, is a novel selective estrogen receptor modulator.

Caffeic acid phenethyl ester (CAPE) is an active ingredient of beehive propolis with a structure similar to phenolic acid. The estrogenic effects of propolis were previously demonstrated through the activation of an estrogen receptor. To identify the estrogenic properties of propolis, CAPE was evaluated using in vitro and in vivo methods. CAPE showed selective binding affinity to human estrogen receptor beta (hERbeta) rather than hERalpha. CAPE also reduced ERalpha expression in MCF-7 and MDA 231 cells. In the yeast estrogen receptor transcription assay, CAPE produced the transcriptional activity of estrogen-responsive element with EC(50) values of 3.72 x 10(-6) M. CAPE did not increase the growth of MCF-7 estrogen receptor-positive breast cancer cells in doses ranging from 10(-7) to 10(-5) M. In order to understand how CAPE acts in animals, CAPE was tested by a uterotrophic bioassay. Treatment with CAPE (100, 500 mg/kg) did not increase the uterine weight relative to 3 microg/kg 17beta-estradiol treatment. The results indicate that CAPE, which is a selective agonist to hERbeta, but does not show any estrogenic effect on estrogen receptor-positive breast cancer cells and in immature rat uterine tissue, is a potential selective estrogen receptor modulator.

Revaprazan prevented indomethacin-induced intestinal damages by enhancing tight junction related mechanisms.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly prescribed medications for alleviating pain and inflammation but may cause gastrointestinal tract damage. Proton pump inhibitors (PPI) prevent NSAID-induced gastric damage but may aggravate intestinal damage via dysbiosis and intestinal permeability alteration. Currently, there is growing interest regarding the influence of potassium competitive acid blockers (PCAB) on NSAID-induced enteropathy. Here, we investigated the relative changes in indomethacin-induced enteropathy by combining indomethacin with pantoprazole (as PPI) or revaprazan (as PCAB). We examined intestinal permeability-related molecular changes in in vitro Caco-2 cell models and in an in vivo indomethacin-induced enteropathy rat model. Indomethacin alone or in combination with pantoprazole significantly increased relative lucifer yellow dye flux and decreased relative trans-epithelial electrical resistance and tight junction protein (TJP) expression compare to normal cells. In contrast, indomethacin combined with revaprazan significantly preserved TJPs compare to indomethacin-treated cells. MLC phosphorylation, Rho activation, and ERK activation responsible for TJP were significantly increased by indomethacin alone or a combination of indomethacin and pantoprazole but not by a combination of indomethacin and revaprazan. Intestinal damage scores significantly increased with indomethacin and pantoprazole combination but not with indomethacin and revaprazan combination. Indomethacin and pantoprazole combination significantly activated Rho-GTPase, p-MLC, and p-ERK but significantly decreased TJP expression. However, indomethacin and revaprazan combination significantly preserved TJPs and inactivated Rho-GTPase, MLC, and ERK. Hence, revaprazan rather than PPIs should be co-administered with NSAIDs to mitigate NSAID-induced intestinal damage.