Misao Yoneda

Effects of Fe3O4 Magnetic Nanoparticles on A549 Cells

Fe3O4 magnetic nanoparticles (MgNPs-Fe3O4) are widely used in medical applications, including magnetic resonance imaging, drug delivery, and in hyperthermia. However, the same properties that aid their utility in the clinic may potentially induce toxicity. Therefore, the purpose of this study was to investigate the cytotoxicity and genotoxicity of MgNPs-Fe3O4 in A549 human lung epithelial cells. MgNPs-Fe3O4 caused cell membrane damage, as assessed by the release of lactate dehydrogenase (LDH), only at a high concentration (100 μg/mL); a lower concentration (10 μg/mL) increased the production of reactive oxygen species, increased oxidative damage to DNA, and decreased the level of reduced glutathione. MgNPs-Fe3O4 caused a dose-dependent increase in the CD44+ fraction of A549 cells. MgNPs-Fe3O4 induced the expression of heme oxygenase-1 at a concentration of 1 μg/mL, and in a dose-dependent manner. Despite these effects, MgNPs-Fe3O4 had minimal effect on cell viability and elicited only a small increase in the number of cells undergoing apoptosis. Together, these data suggest that MgNPs-Fe3O4 exert little or no cytotoxicity until a high exposure level (100 μg/mL) is reached. This dissociation between elevated indices of cell damage and a small effect on cell viability warrants further study.

Morphological characteristics of basal-like subtype of breast carcinoma with special reference to cytopathological features

Expression profiling of invasive breast carcinomas by DNA microarray techniques has identified five distinct subtypes of tumors (luminal A, luminal B, normal breast-like, HER2 overexpression, and basal-like) that are associated with different clinical outcomes and with different chemotherapy. Basal-like carcinoma is associated with younger patient age, high histological grade, aggressive clinical course, development of distant metastasis, poor prognosis, and relatively high mortality rate. Basal-like carcinomas do not express estrogen receptor, progesterone receptor, or HER2 (triple-negative phenotype). Therefore, patients with basal-like carcinomas are not likely to benefit from endocrine therapies or trastuzumab, but are likely to benefit from systemic chemotherapy. Although genetic, morphological, and immunohistochemical features of basal-like carcinomas have been reported, there is no universal definition for those tumors. Furthermore, there are no specific morphological and immunohistochemical features that can identify those tumors in routine diagnostic materials. In the present paper, we present data of histological and cytological features of basal-like breast carcinoma, and discuss about its morphological spectrum.

The expression and function of Toll-like receptors 3 and 9 in human colon carcinoma

Toll-like receptors (TLRs) are pattern-recognition receptors that are important in immune signaling. TLR recognition of various viral components including double-stranded RNA (TLR3) and unmethylated CpG-DNA (TLR9) plays a crucial role in cell survival. However, TLR expression and function in colon carcinoma cells are not well clarified. We investigated the expression of TLR3 and TLR9 in colon carcinoma cells using immunohistochemical methods. The function of TLR3 and TLR9 signaling in carcinoma cell lines was studied by direct cell stimulation with, or by cell transfection of, polyinosinic-polycytidylic acid (Poly I:C), a synthetic form of dsRNA, and by cell stimulation with CpG-oligodeoxynucleotides (ODNs), respectively. Positive TLR3 and TLR9 immunohistochemical staining was observed in 91 and 86% of human hepatocellular carcinoma (HCC) tissues, respectively. Cell surface stimulation of TLR3 with Poly I:C did not affect cell viability but it did activate NF-κB activity. By contrast, stimulation of intracellular TLRs with transfected Poly I:C significantly induced apoptosis. Cell surface stimulation of TLR9 with CpG-ODNs promoted cell proliferation, and, furthermore, these CpG-ODN TLR9 agonists reduced the cytotoxicity of the anticancer drug adriamycin. Cell surface expression of TLR3 and TLR9 in colon carcinoma cells plays an important role in cell survival. In addition, the proapoptotic activity of intracellularly expressed TLR3 may provide the possibility of using TLR3 agonists as novel clinical cytotoxic agents against colon carcinoma cells.

High level expression of STAG1/PMEPA1 in an androgen-independent prostate cancer PC3 subclone

In this paper, we describe the isolation and characterization of two PC3 subclones. One subclone, mr, showed an epithelial phenotype, the other, M1, showed a sarcomatous morphology. Transplanted into nude mice, mr developed tumors at a dramatically faster rate than M1. Comparing the two subclones, differentially expressed genes were identified, including E-cadherin, IL-8 and STAG1/PMEPA1. These genes were expressed at higher levels in mr than in M1.

Significant intratumoral heterogeneity of human epidermal growth factor receptor 2 status in gastric cancer: A comparative study of immunohistochemistry, FISH, and dual-color in situ hybridization.

The assessment of human epidermal growth factor receptor 2 (HER2) status is crucial for selecting patients with gastric cancer who may benefit from HER2‐targeted therapy. Accurate assessment using biopsy specimens is important for patients with advanced‐stage cancer. Intratumoral heterogeneity of HER2, however, is a major challenge in HER2 testing. Here, we aimed to examine whether assessment of HER2 status could be accurately carried out with currently used methods, namely, immunohistochemistry (IHC), FISH, and dual‐color in situ hybridization (DISH). Human epidermal growth factor receptor 2 status was evaluated in 108 biopsy tissues from patients with gastric adenocarcinoma and 70 matched surgical specimens by IHC, FISH, and DISH; HER2 amplification was detected in 11 (10.2%) out of 108 biopsy specimens. The IHC and FISH results were well correlated, and FISH and DISH results were consistent for all cases. The overall concordance rate of HER2 status between biopsy tissues and surgical specimens was 91.4%. All six discordant cases were false negative on biopsy; of these cases, five showed HER2 heterogeneity on surgical resection. Assessment of the HER2 status of biopsy tissues could predict the status of the whole tumor; however, a proportion of these cases may be discordant because of intratumoral heterogeneity.

Human Equilibrative Nucleoside Transporter 1 Expression in Endoscopic Ultrasonography-Guided Fine-Needle Aspiration Biopsy Samples Is a Strong Predictor of Clinical Response and Survival in the Patients With Pancreatic Ductal Adenocarcinoma Undergoing Gemcitabine-Based Chemoradiotherapy.

ObjectivesThis study aimed to clarify whether pretreatment human equilibrative nucleoside transporter (hENT1) expressions in endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) specimens obtained from resectable, borderline resectable, and locally advanced unresectable pancreatic ductal adenocarcinoma (PDAC) are concordant with those in the resected specimen after gemcitabine-based chemoradiotherapy (Gem-CRT) and to validate the utility of hENT1 expression using EUS-FNAB samples as a prognostic marker. MethodsWe evaluated the relationship between hENT1 expressions assessed by immunohistochemical staining and clinical outcomes in 51 of 76 patients with PDAC who were diagnosed by EUS-FNAB and received preoperative Gem-CRT. ResultsThe concordance rate of hENT1 expressions was 89.2% (K = 0.681). Median survival time (month) in the 51 whole patients and 37 patients with resection was significantly longer in hENT1 positive than in hENT1 negative: 25.0 and 30.0 versus 9.0 and 9.0, respectively. A multivariate analysis confirmed that hENT1 expression was an independent prognostic factor in both whole patients and those with resection. Regardless of T3 and T4, hENT1-positive patients with resection had significantly better prognosis than hENT1-negative patients, whose prognosis was similar to those without resection. ConclusionsThe assessment of hENT1 expression using EUS-FNAB samples before Gem-CRT provides important information on patients with PDAC who can benefit from curative-intent resection.

Human β-defensin-3 inhibits migration of colon cancer cells via downregulation of metastasis-associated 1 family, member 2 expression. Corrigendum in /ijo/46/4/1858

The innate immune system plays an important role as the first line of defense against many types of microbes. Accumulating reports suggest that human β-defensins (hBDs) are expressed by and have certain roles in some cancer cells. In this study, we investigated the roles of hBD-3 in colon cancer cells. The expression of hBD-3 was examined by reverse transcriptase-polymerase chain reaction analysis of colon cancer cell lines and immunohistochemical staining of colon cancer tissues. The effect of hBD-3 on proliferation of colon cancer was assessed using the MTT assay and a real-time cell analyzer, and the effect of hBD-3 on the migration of colon cancer cells was also examined. The results showed that hBD-3 is not expressed in colon cancer cells but is produced by tumor-infiltrating monocytes. Migration of colon cancer cells was significantly inhibited by hBD-3 in a dose-dependent manner, although proliferation of colon cancer cells was not affected by administration of hBD-3. Moreover, reduced expression of metastasis-associated 1 family, member 2 (MTA2) mRNA in colon cancer cells was associated with exposure to hBD-3. In conclusion, progression of colon cancer was inhibited by hBD-3 in a paracrine fashion. Therefore, hBD-3 may be a potent new agent for treating colon cancer.

Resveratrol sensitizes HepG2 cells to TRAIL-induced apoptosis.

Resveratrol is a natural polyphenol found in a wide variety of plants, including grapes, berries, and peanuts. Resveratrol can modulate a wide spectrum of molecular targets, including those involved in cancer signaling pathways. Here, we evaluated the role of resveratrol in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and examined the molecular mechanisms in the human hepatocellular carcinoma cell line HepG2. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to assess cell viability, flow cytometry to analyze cell cycle and apoptosis, and immunoblotting to detect protein expression. Resveratrol decreased cell viability at a concentration of 100 &mgr;mol/l or higher. At a concentration of 50 &mgr;mol/l, resveratrol induced S phase arrest of the cell cycle without apoptosis. In addition, phospho-AMPK increased significantly in a dose-dependent manner. Resveratrol was found to synergistically augment TRAIL-induced apoptosis. The rates of early apoptosis were 3.4, 9.6, and 49.6% on treatment with 50 &mgr;mol/l resveratrol, 10 ng/ml TRAIL, and both reagents, respectively. Resveratrol significantly downregulated the expression of survivin in a dose-dependent manner. In conclusion, we found that that resveratrol could augment TRAIL sensitivity by downregulating survivin. These results suggest that combination resveratrol with TRAIL may be an effective new strategy for the treatment of hepatocellular carcinoma.

Multicellular Spheroid Culture Models: Applications in prostate Cancer Research and Therapeutics

Prostate cancer is one of the most prevalent cancers in men in Western countries, increasing in frequency with age through the most advanced years. Patients with localized prostate cancer are generally treated with radical prostectomy or radiation therapy. However, treatment of more malignant stages of the disease is problematic. Docetaxel-based chemotherapy in men with androgen-independent prostate cancer has been shown to have survival benefits but hormonal manipulation and other chemotherapeutic regimens, especially for androgen-independent lesions, have uncertain value. While research into the complex pathophysiology of advanced prostate cancer has led to identification of mechanisms and target molecules, it nevertheless remains necessary to develop new anticancer drugs. Cell culture models that mimic the structure and features of prostate cancer in vivo are necessary for research on tumor biology and design of novel anticancer therapies. In this context, 3-dimensional cultures of prostate cancer cells, including multicellular spheroid (MCS) cultures, started attracting increasing attention. The present review provides up-to-date information regarding the significance of MCS culture for identification of mechanisms underlying human malignancies, including prostate cancer, and possible targets for prostate cancer therapies.

Effects of Mycobacteria Major Secretion Protein, Ag85B, on Allergic Inflammation in the Lung

Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B), which is one of the major protein antigens secreted from M. tuberculosis. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.