Yi Rang Na

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

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.

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.

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.

Male-specific hepatitis B virus large surface protein variant W4P potentiates tumorigenicity and induces gender disparity

BackgroundThe underlying mechanisms of carcinogenesis and gender disparity in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remain unclear. Recently, we reported a novel HCC-related W4P/R mutation in the large surface protein (LHB) of HBV genotype C, which was found exclusively in male HCC patients.MethodsLHB sequences from a carrier (wild type; WT) and W4P variant LHB sequence from an HCC patient were cloned and used to generate NIH3T3 and Huh7 cell lines. Cell proliferation and in vitro tumorigenicity were assessed by cell growth and transformation assays. Male and female nude mice were injected with the cells to determine in vivo tumorigenicity. To confirm the effect of estrogen in W4P-mediated tumorigenicity, male mice were injected with estrogen and challenged with W4P-expressing cells. The serum levels of different cytokines from the mouse model and patients were analyzed by ELISA. A critical role of interleukin (IL)-6 signaling in W4P-mediated tumorigenicity was tested by inhibition of Jak2.ResultsAlthough both WT and W4P variant LHBs enhanced cell proliferation by regulating the cell cycle and facilitated cell colony formation, the W4P variant demonstrated significantly higher activity. NIH3T3 cells expressing variant LHB, but not the WT, induced tumor in a nude mouse model. Tumor masses produced by variant LHB were significantly larger in male than female mice, and significantly reduced by estrogen. IL-6, but not tumor necrosis factor-α, was elevated in male mice harboring W4P-induced tumor, and was reduced by estrogen. IL-6 levels of HCC patients with the W4P variant were significantly higher than those of patients with WT LHB. W4P LHB induced higher production of IL-6 than WT LHB in cell lines, and the level was reduced by estrogen. The ability to reduce cell proliferation and colony formation of W4P LHB was hampered by inhibition of IL-6 signaling.ConclusionsThis study suggests that the W4P mutation during the natural course of chronic hepatitis B infection may contribute to HCC development, particularly in male patients, in an IL-6-dependent manner.

Interleukin-6-induced Twist and N-cadherin enhance melanoma cell metastasis.

Melanoma patients frequently have elevated serum levels of interleukin-6 (IL-6), which is correlated with a poor prognosis. IL-6 activates STAT3 phosphorylation, inducing the transcription of genes that regulate tumor cell proliferation and antiapoptosis. In addition, recent evidence suggests that IL-6 induces the epithelial-to-mesenchymal transition and enhances the invasiveness of tumor cells of epithelial origin. However, it is unknown whether IL-6 affects mesenchymal tumor cells. In this study, we examined the effects of IL-6 on melanoma cells and found that IL-6 can enhance their metastatic potential by regulating the expression of Twist and N-cadherin. First, we confirmed that human melanoma tissues express IL-6 (especially at the lesion site), the IL-6 receptor, N-cadherin, and nuclear Twist. Next, we found that IL-6 induces STAT3 phosphorylation in WM-266-4 human melanoma cells, resulting in transient upregulation of Twist, which is a key regulator of metastasis. Importantly, the expression of N-cadherin, a protein downstream of Twist, was also increased on the cell surface after treatment with IL-6. These cells showed enhanced invasiveness, assessed using an invasion assay, and formed more metastatic nodules in the lungs of NOD-SCID mice after an intravenous injection. Importantly, melanoma cells with knocked-down N-cadherin formed less lung nodules compared with control in the NOD-SCID mouse model. Our data suggest that increased serum IL-6 in cancer patients could increase the invasiveness of melanoma cells and accelerate metastasis. Blocking IL-6 in the melanoma microenvironment may therefore inhibit disease progression.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up‐regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 × 10−6, 40 × 10−6 and 80 × 10−6 m of benomyl‐treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase‐based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 × 10−6 m concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds. Copyright

Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs

BackgroundWith high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs.MethodsWe injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer’s solution containing carboplatin and melphalan after the injection of retinoblastoma cells.ResultsThe degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP.ConclusionsThis orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.