Exosomes derived from cancerous and non-cancerous cells regulate the anti-tumor response in the tumor microenvironment.

The tumor microenvironment (TME) is a unique platform of cancer biology that considers the local cellular environment in which a tumor exists. Increasing evidence points to the TME as crucial for either promoting immune tumor rejection or protecting the tumor. The TME includes surrounding blood vessels, the extracellular matrix (ECM), a variety of immune and regulatory cells, and signaling factors. Exosomes have emerged to be molecular contributors in cancer biology, and to modulate and affect the constituents of the TME. Exosomes are small (40-150 nm) membrane vesicles that are derived from an endocytic nature and are later excreted by cells. Depending on the cells from which they originate, exosomes can play a role in tumor suppression or tumor progression. Tumor-derived exosomes (TDEs) have their own unique phenotypic functions. Evidence points to TDEs as key players involved in tumor growth, tumorigenesis, angiogenesis, dysregulation of immune cells and immune escape, metastasis, and resistance to therapies, as well as in promoting anti-tumor response. General exosomes, TDEs, and their influence on the TME are an area of promising research that may provide potential biomarkers for therapy, potentiation of anti-tumor response, development of exosome-based vaccines, and exosome-derived nanocarriers for drugs.

Regulation of p53 during senescence in normal human keratinocytes.

p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16(INK4A) , induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells.

Development of oral osteomucosal tissue constructs in vitro and localization of fluorescently-labeled bisphosphonates to hard and soft tissue.

Bisphosphonates (BPs) are anti-resorptive agents commonly used to treat bone-related diseases; however, soft tissue-related side-effects are frequently reported in some BP users, such as oral or gastrointestinal (GI) ulcerations. BPs are stable analogs of pyrophosphate and have high affinity to hydroxyapatite, allowing them to bind to the bone surfaces and exert suppressive effects on osteoclast functions. However, the underlying mechanisms as to how bone-seeking BPs also exert cytotoxic effects on soft tissue remain unknown. In the present study, we investigated the localization of nitrogen-containing BPs (N-BPs) in hard and soft tissue using fluorescently-labeled N-BPs in vitro. We developed osteomucosal tissue constructs in vitro to recapitulate the hard and soft tissue of the oral cavity. A histological examination of the osteomucosal tissue constructs revealed a differentiated epithelium over the bone containing osteocytes and the periosteum, similar to that observed in the rat palatal tissues. Following treatment with the fluorescently-labeled bisphosphonate, AF647-ZOL, the osteomucosal constructs exhibited fluorescent signals, not only in the bone, but also in the epithelium. No fluorescent signals were observed from the control- or ZOL-treated constructs, as expected. Collectively, the data from the present study suggest that N-BPs localize to epithelial tissue and that such a localization and subsequent toxicity of N-BPs may be associated, at least in part, with soft tissue-related side-effects.

Camphorquinone inhibits odontogenic differentiation of dental pulp cells and triggers release of inflammatory cytokines.

INTRODUCTION: Camphorquinone (CQ) is a photoinitiator that triggers polymerization of light-curing materials such as dental adhesives and composites. CQ does not become a part of the polymer network, suggesting that CQ can be leached out into surrounding environment including dental pulp and exert adversary effects on tissues. In order to understand the mechanisms of CQ-induced side effects, we investigated the effect of CQ on cell viability, cytokine secretion, and odontogenic differentiation of dental pulp stem cells in vitro. METHODS: Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after CQ exposure. Western blotting was performed for p16(INK4A), p21(WAF1), and p53. Secretory cytokines were evaluated using the membrane-enzyme-linked immunosorbent assay as well as conventional and quantitative reverse-transcription polymerase chain reaction. The effects of CQ on odontogenic differentiation were evaluated using alkaline phosphatase and alizarin red S staining methods. RESULTS: CQ treatment suppressed the proliferation of DPSCs and induced the expression of p16(INK4A), p21(WAF1), and p53. Levels of proinflammatory cytokines (eg, interleukin 6, interleukin 8, and matrix metalloproteinase-3 [MMP3]) were increased by CQ treatment. CQ also inhibited odontogenic differentiation and mineralization capacities of DPSC and MC3T3-E1 cells. CONCLUSIONS: Our study showed that CQ may trigger pulpal inflammation by inducing proinflammatory cytokine production from the pulpal cells and may impair odontogenic differentiation of dental pulp cells, resulting in pulpal irritation and inflammation.

Identification of senescence-inducing microRNAs in normal human keratinocytes.

MicroRNAs (miRNAs) are epigenetic regulators of eukaryotic gene expression and play key roles in many cellular processes. However, the role of miRNAs for replicative senescence of normal human keratinocytes (NHKs) remains unknown. Thus, we examined the expression profiles of 847 miRNAs in exponentially replicating and senescent NHKs and identified 126 senescence-associated miRNAs (SA-miRs). Among SA-miRs, 117 miRNAs (93%) were upregulated and 9 miRNAs (7%) were downregulated in senescent NHKs compared to those of exponentially replicating cells. Among the above miRNAs, we selected two miRNAs, miR-137 and miR-668, for further investigation because they were consistently upregulated with replicative senescence of three independent NHK cultures. Ectopic overexpression of miR-137 or miR-668 induced senescence in rapidly proliferating NHKs; a notable increase in senescence-associated ß-galactosidase activity, p16INK4A and p53 was observed, indicating that they are novel senescence-inducing miRNAs. In addition, these senescence-inducing miRNAs were gradually increased during organismal aging of normal human oral epithelia. We also detected downregulation of miR-137 and miR-668 in many tested human head and neck squamous cell carcinoma cell lines. Since senescence would be viewed as a potent tumor suppressive pathway, the newly identified senescence-inducing miRNAs deserve to be further investigated for their therapeutic application in cancer treatment.

miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras.

MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3'-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.

Immune blood biomarkers of Alzheimer disease patients.

Alzheimer disease (AD) patients have an impairment of anti-amyloid-beta (Abeta) innate immunity and a defect in immune gene transcription [Fiala, M., Liu, P.T., Espinosa-Jeffrey, A., Rosenthal, M.J., Bernard, G., Ringman, J.M., Sayre, J., Zhang, L., Zaghi, J., Dejbakhsh, S., Chiang, B., Hui, J., Mahanian, M., Baghaee, A., Hong, P., Cashman, J., 2007b. Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin. Proc. Natl. Acad. Sci. U. S. A. 104, 12849-12854]. Early diagnosis is a cornerstone of preventive approaches to AD. Phospho-tau and Abeta CSF levels are useful markers of neurodegeneration but not of a process leading to neurodegeneration. To detect an early biomarker of AD, we developed a flow cytometric test of Abeta phagocytosis, which was 94% positive (<400 MFI units) in AD patients (mean age+/-SEM 77+2.2 years; mean score+/-SEM 198.6+/-25.5 MFI units) and 60% positive in MCI patients (77+/-5.6 years; 301+/-106 MFI units). Control subjects, active senior university professors, were 100% negative (74.2+/-4.2 years; 1348+/-174 MFI units). The test had a low specificity in older caregivers and older amyotrophic lateral sclerosis (ALS) patients. We also tested transcriptional regulation of the genes MGAT-III and Toll-like receptor-3 in macrophages. Macrophages of "Type I" patients (a majority of patients) showed gene down regulation at baseline and up regulation by curcuminoids; macrophages of "Type II" patients showed opposite responses. The results of flow cytometric testing suggest that normal Abeta phagocytosis is associated with healthy cognition and lesser risk of AD. The significance of abnormal results in aged persons should be investigated by prospective studies to determine the risk of AD.