Seung-Hyeok Seok

Cyclooxygenase-2 Inhibition Blocks M2 Macrophage Differentiation and Suppresses Metastasis in Murine Breast Cancer Model

Tumor cells are often associated with abundant macrophages that resemble the alternatively activated M2 subset. Tumor-associated macrophages (TAMs) inhibit anti-tumor immune responses and promote metastasis. Cyclooxygenase-2 (COX-2) inhibition is known to prevent breast cancer metastasis. This study hypothesized that COX-2 inhibition affects TAM characteristics potentially relevant to tumor cell metastasis. We found that the specific COX-2 inhibitor, etodolac, inhibited human M2 macrophage differentiation, as determined by decreased CD14 and CD163 expressions and increased TNFα production. Several key metastasis-related mediators, such as vascular endothelial growth factor-A, vascular endothelial growth factor-C, and matrix metalloproteinase-9, were inhibited in the presence of etodolac as compared to untreated M2 macrophages. Murine bone marrow derived M2 macrophages also showed enhanced surface MHCII IA/IE and CD80, CD86 expressions together with enhanced TNFα expressions with etodolac treatment during differentiation. Using a BALB/c breast cancer model, we found that etodolac significantly reduced lung metastasis, possibly due to macrophages expressing increased IA/IE and TNFα, but decreased M2 macrophage-related genes expressions (Ym1, TGFβ). In conclusion, COX-2 inhibition caused loss of the M2 macrophage characteristics of TAMs and may assist prevention of breast cancer metastasis.

Bone morphogenetic protein 7 induces mesenchymal-to-epithelial transition in melanoma cells, leading to inhibition of metastasis

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial‐to‐mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial‐to‐mesenchymal transition and its reversed process mesenchymal‐to‐epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM‐266‐4 melanoma cell growth and metastasis. An MTT assay was conducted in WM‐266‐4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT‐PCR was used to determine MET in morphologically changed BMP7‐treated melanoma cells. MET‐induced cells expressed less a basic helix‐loop‐helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7‐induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM‐266‐4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells. (Cancer Sci 2009)

Rat pancreatitis produced by 13-week administration of zinc oxide nanoparticles: biopersistence of nanoparticles and possible solutions

Zinc oxide (ZnO) nanoparticles (NPs) are used in diverse applications ranging from paints and cosmetics to biomedicine and food. Although micron‐sized ZnO is a traditional food supplement, ZnO NPs are an unknown public health risk because of their unique physicochemical properties. Herein, we studied the 13‐week subchronic toxicity of ZnO NPs administered via the oral route according to Organization for Economic Cooperation and Development (OECD) test guideline 408. Well‐dispersed ZnO NPs were administered to Sprague–Dawley (SD) rats (11/sex/group) at doses of 67.1, 134.2, 268.4 or 536.8 mg kg–1 per body weight over a 13‐week period. The mean body weight gain in males given 536.8 mg kg–1 ZnO NPs was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg kg–1 ZnO NPs had significant changes in anemia‐related hematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg kg–1, whereas no histological changes were observed in the other treatment groups. To evaluate the mechanism of toxicity, we performed a bio‐persistence study and evaluated the effects of the ZnO NPs on cell proliferation. The treatment of a human gastric adenocarcinoma cell line with ZnO NPs resulted in a significant inhibition of cellular proliferation. The anti‐proliferative effect of ZnO NPs or Zn2+ was effectively blocked by treatment with chelators. These results indicate that the bio‐persistence of ZnO NPs after ingestion is key to their toxicity; the no‐observed‐adverse effect level (NOAEL) of ZnO NPs was found to be 268.4 mg kg–1 per day for both sexes. Copyright

A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test.

Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method.

Developmental toxicity and brain aromatase induction by high genistein concentrations in zebrafish embryos

Genistein is a phytoestrogen found at a high level in soybeans. In vitro and in vivo studies showed that high concentrations of genistein caused toxic effects. This study was designed to test the feasibility of zebrafish embryos for evaluating developmental toxicity and estrogenic potential of high genistein concentrations. The zebrafish embryos at 24 h post-fertilization were exposed to genistein (1 3 10−4 M, 0.5 3 10−4 M, 0.25 3 10−4 M) or vehicle (ethanol, 0.1%) for 60 h. Genistein-treated embryos showed decreased heart rates, retarded hatching times, decreased body length, and increased mortality in a dose-dependent manner. After 0.25 3 10−4 M genistein treatment, malformations of survived embryos such as pericardial edema, yolk sac edema, and spinal kyphosis were also observed. TUNEL assay results showed apoptotic DNA fragments in brain. This study also confirmed the estrogenic potential of genistein by EGFP expression in the brain of the mosaic reporter zebrafish embryos. This study first demonstrated that high concentrations of genistein caused a teratogenic effect on zebrafish embryos and confirmed the estrogenic potential of genistein in mosaic reporter zebrafish embryos.

Terahertz dynamic imaging of skin drug absorption

Terahertz (THz) imaging is a nondestructive, label-free, rapid imaging technique which gives the possibility of a real-time tracing of drugs within the skin. We evaluated the feasibility of THz dynamic imaging for visualizing serial changes in the distribution and penetration of a topical agent, dimethyl sulfoxide (DMSO) containing ketoprofen, using excised mouse skins. THz imaging was performed for 6 h after drug application to the skin and was compared with the results obtained using the Franz cell diffusion test, a standard in vitro skin absorption test. THz dynamic reflection imaging showed that the reflection signals decreased rapidly during the early time period, and remained constant through the late time period. The area of drug permeation within the skin layer on THz imaging increased with time. The dynamic pattern of THz reflection signal decrease was similar to that of DMSO absorption analyzed by the Franz cell diffusion test, which indicates that THz imaging mainly reflects the DMSO component. This study demonstrates that THz imaging technique can be used for imaging the spatial distribution and penetration of drug-applied sites.

Isolation and characterization of spheroid cells from human malignant melanoma cell line WM-266-4.

Background/Aims: Spheroid cells which can grow as nonattached spheroids in vitro culture condition are considered as tumor-initiating cells that have properties similar to those of stem cells. However, the existence of spheroid cells in WM-266-4, a human malignant metastatic melanoma cell line, has not yet been reported. Methods: Accordingly, we investigated whether WM-266-4 can form spheroids, and characterized these spheroids using qRT-PCR, histology, immunohistochemistry and xenograft. Results: WM-266-4 contains a small subpopulation of cells that grow as spheroids and express genes strongly related to tumor malignancy and stem-like factors. Second, histological analysis of the spheres revealed that they consist of 300–400 round cells per sphere with a high karyoplasmic ratio. They have a basophilic cytoplasm and are highly pleomorphic in size, and sometimes multinucleated and giant. Third, although there were differences between the spheroid and bulk cells, they both have high tumorigenic potential, as both cell types formed a tumor mass upon injection of only 100 cells in nude mice. Conclusion: We characterized the spheroid cells in an established melanoma cell line. We suggest that enriched spheroid cells might contain more dedifferentiated progenitor cells, but we could not conclude spheroid cells are cancer stem cells.

KeraSkin-VM: a novel reconstructed human epidermis model for skin irritation tests.

Several alternative in vitro methods to evaluate skin irritants have been developed recently. In July 2010, OECD officially endorsed the validated reference method (VRM) that uses reconstituted human epidermis (RhE) models as replacements for the in vivo skin irritation test. This study evaluated the KeraSkin-VM model, a novel human epidermis model that was reconstructed with Asian skin tissue using 20 reference chemicals according to the OECD TG 439 performance standard. The test chemicals were applied to the epidermal surface side for 45 min and then rinsed, and then incubated for 42 h post-treatment. An overall accuracy of 80%, sensitivity of 90% and specificity of 70% were obtained when the results from KeraSkin-VM were compared with UN GHS categories, which was comparable to the EpiDerm Skin irritation test (SIT) rates. Furthermore, KeraSkin-VM demonstrated good performance in terms of within-laboratory reproducibility and predictive capacity to screen skin irritants.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

Background: Aggressive breast tumor cells secrete platelet-derived growth factor C (PDGF-C). Results: PDGF-C prevents staurosporine-induced macrophage apoptosis through PI3K/Akt activation and Bad phosphorylation, resulting in the inhibition of caspase activation and PARP cleavage. Conclusion: PDGF-C has an anti-apoptotic effect on macrophages. Significance: PDGF-C secreted from malignant tumor cells could affect the survival of tumor-associated macrophages. PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

Inhibition of lytic reactivation of Kaposi's sarcoma― associated herpesvirus by alloferon

BACKGROUND Alloferon, an immunomodulatory peptide, has antiviral capability against herpesvirus. In this research, we aimed to investigate the effect of alloferon on the regulation of the life cycle of Kaposis sarcoma-associated herpesvirus (KSHV), and its mechanisms. We also assessed the antiviral activity of alloferon on natural killer (NK) cells as an early antiviral immune responder. METHODS We first examined the change in cell proliferation and the expression of the viral genes in a KSHV-infected cell line, body-cavity-based B lymphoma (BCBL)-1, under the lytic cycle by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment. To elucidate the antiviral mechanism of alloferon, we tested calcium influx and the activation of the extracellular signal-regulated kinase (ERK) pathway. Furthermore, we evaluated the cytotoxicity of NK cells against BCBL-1 by alloferon. RESULTS Alloferon effectively recovered the suppressed proliferation of BCBL-1 by TPA, which was achieved by the down-regulation of lytic-cycle-related viral genes, RTA, K8 and vIRF2. To clarify the signal transduction pathways related to the regulation of the viral genes by alloferon, we confirmed that the calcium influx into BCBL-1 was apparently inhibited by alloferon, which preceded the suppression of the phosphorylation of ERK and the activation of AP-1 by TPA. Moreover, when NK cells were exposed to alloferon, their cytolytic activity was improved, and this was mediated by the enhancement of perforin/granzyme secretion. CONCLUSIONS The results of this study suggest that alloferon can be used as an effective antiviral agent for the regulation of the KSHV life cycle by the down-regulation of AP-1 activity and for the the enhancement of antiviral immunity by up-regulation of NK cell cytotoxicity.