The combination of DHEA, histamine, and insulin increases adipogenic differentiation and enhances tissue transplantation outcome in mice.

Adipose stem cells (ASCs) are pluripotent cells that can generate pure fat tissue for regeneration. Differentiated adipose cells have been generated by a common inducer cocktail composed of dexamethasone, insulin, and isobutylmethylxanthine (DIM). The major drawbacks of adipose cells are their tendency to float on the culture media and their cost. To overcome some of these disadvantages, a new inducer cocktail that includes insulin, dehydroepiandrosterone, and histamine (DH IH) was tested. As a result, lipid accumulation was elevated more than twofold with DH IH than with DIM. Cell adhesion and viability, which are important factors for stable differentiation, were increased with DH IH and were proven through measurement of mRNA expression levels of adhesion marker genes, N-cadherin and vascular cell adhesion molecule, as well as through an alamar blue assay. The expression of adipogenesis-related genes, adiponectin, and glucose transporter type 4 lasted for a long time. To improve the efficiency of grafting, cell adhesion and neovascularization need to be increased. Neovascularization was observed around the transplanted adipose cells, which showed a higher number of vessel formation in DH IH than in DIM. The above results suggest that DH IH can produce pure differentiated adipose cells effectively and enhance their adhesion onto the target location when these differentiated adipose cells were applied as a clinical resource.

Interleukin-18-mediated interferon-gamma secretion is regulated by thymosin beta 4 in human NK cells.

Thymosin beta 4 (Tß4) is the major G-actin sequestering molecule and is abundant in lymphoid tissues. However, it is not clear what regulates Tß4 expression and what its function is on natural killer (NK) cells. We investigated whether interleukin-18 (IL-18) has a role in Tß4 expression and if enhanced Tß4 influences IL-18-mediated interferon-gamma (IFN-γ) secretion. In this study, recombinant human IL-18 (rhIL-18) enhanced the endogenous level of Tß4 through p38MAPK and JNK signaling pathway in the human NK cell line, NK-92MI. Overexpression of endogeneous Tß4 stimulated IFN-γ expression and secretion. Additionally, pretreatment with an inhibitor for Tß4 decreased IL-18-enhanced IFN-γ secretion, and transfection with Tß4-specific short hairpin RNA resulted in reduction of IFN-γ production in primary NK cells as well as in the human NK cell line. Taken together, these data indicated that Tß4 is regulated by IL-18 and is involved in IL-18-enhanced IFN-γ secretion in NK cells.

Erythroid differentiation regulator 1, an interleukin 18-regulated gene, acts as a metastasis suppressor in melanoma.

Erythroid differentiation regulator (Erdr1) was first discovered in mouse leukemia cell lines and functions as a stress-related survival factor. This study investigated whether Erdr1 regulates murine melanoma progression, as well as the mechanism involved in Erdr1-regulated metastasis. The expression of Erdr1 is negatively correlated with IL-18 expression, which has a pro-cancer effect in melanoma. To study the role of Erdr1 as an anti-cancer factor, cell migration, invasion, and proliferation were measured. Erdr1 overexpression markedly inhibited the level of cell migration, invasion, and proliferation in B16F10 cells in vitro. In addition, Erdr1 overexpression significantly suppressed melanoma lung colonization, metastasis, and tumor growth in vivo. To identify the factors involved in Erdr1-reduced metastasis, heat shock protein 90 (HSP90), a well-known stress protein and contributor to tumor metastasis, was examined. We found that HSP90 was significantly decreased in Erdr1-overexpressing cells. Functional analysis demonstrated that HSP90 small-interfering RNA transfection reduced the migration ability and metastasis of melanoma. In conclusion, Erdr1 shows a powerful anti-metastasis effect that leads to the ability to reduce the metastatic potential of murine malignant melanoma cells. Erdr1 is an anti-metastatic factor that may be a possible therapeutic target for treatment of melanoma.

Thymosin ß4 expression correlates with lymph node metastasis through hypoxia inducible factor-α induction in breast cancer.

Intratumoral hypoxia has been correlated with distant metastatic potential. Two hypoxia inducible factors (HIFs), HIF-1α and HIF-2α, are induced by hypoxia, and high expression of these proteins has been correlated to angiogenesis and distant metastasis. Thymosin ß4 (Tß4) is frequently highly expressed in cancer, and this overexpression correlates with malignant progression. The objective of this study was to investigate the clinical correlation of HIF-α with Tß4 and the intracellular functional roles of Tß4 on HIF-α activation. We examined HIF-1α, HIF-2α and Tß4 expressions in clinical human breast carcinoma (n=70) by immunohistochemistry. We show that high expression of HIF-1α and HIF-2α strongly correlates with Tß4 expression (P≤0.0001) and overexpression of Tß4 correlates significantly with patients with lymph node metastasis (P<0.05) of human breast cancer. Additionally, we demonstrate that hypoxia up-regulates intracellular Tß4 protein, which then affects HIF-α activity, which is the key in regulating VEGF expression. We confirmed that hypoxia-induced intracellular Tß4 and HIF-α activities were reduced by interference of Tß4 expression using Tß4 shRNA lentivirus. Vascular epidermal growth factor (VEGF)-A, a well-recognized lymphangiogenic cytokine, was also down-regulated, but VEGF-C and VEGF-D expressions were not affected. These findings suggest that the overexpression of Tß4 is strongly associated with HIF-1α and HIF-2α expression and is also clinicopathologically involved with lymph node metastatic potential of breast cancer through the modulation of HIF-αactivation and induction of VEGF-A. Ultimately, these results highlight Tß4 as a potentially therapeutic target in malignant cancers.

Botulinum toxin enhances the implantation effect of adipocytes in C57/BL6 mice.

BACKGROUND: Recently, many plastic surgeons have been using adipogenic-differentiated cell implantation for remodeling scars in patients. However, this technique is not a long-term solution because implanted cells disappear gradually. Therefore, we investigated a method to increase the grafted cell preservation rate by using an effective adjuvant, botulinum toxin. METHODS: The adipogenic-differentiated cells were subcutaneously injected in the dorsal area of C57/BL6 mice with or without botulinum toxin. Two and six weeks later we analyzed the residual volume and confirmed the characteristics of the implanted cells by real-time RT-PCR and immunohistochemistry. RESULTS: Two and six weeks after transplantation we found that the residual volume of the transplantation site was higher in the botulinum toxin-treated group than in the untreated group. We also confirmed that the residual transplanted area has characteristics of adipogenic tissue by histological analysis. Next, to determine the mechanism related to the enhanced preservation rate of grafted cells via treatment with botulinum toxin, we performed immunohistochemical staining for the angiogenesis-related marker CD31. We found that CD31 expression was higher in the botulinum toxin-treated group than in the untreated group. CONCLUSION: We have shown that in vivo grafted adipocyte cell preservation can be enhanced by treatment with botulinum toxin as an adjuvant. We suggest that botulinum toxin further increases this graft preservation rate by enhancing angiogenesis.

Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells.

Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-gamma inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-gamma production, we measured IL-18-induced IFN-gamma production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-gamma expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-gamma production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-gamma production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-gamma production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-gamma production via p38 MAPK.

Elevated peroxiredoxin 1, but not NF-E2-related factor 2, is an independent prognostic factor for disease recurrence and reduced survival in stage I non-small cell lung cancer.

PURPOSE: Lung cancer is the leading cause of cancer death with chance of survival restricted to a subset of non-small cell lung cancer (NSCLC) patients able to undergo surgical resection. However, the recurrence rate of NSCLC after surgery remains high with few prognostic indicators of clinical outcome. Peroxiredoxin1 (Prx1) is shown to be elevated in various cancers and confers an aggressive survival phenotype. We recently cloned the prx1 promoter and found that NF-E2-related factor 2 (Nrf2) is a key transcription factor for prx1 up-regulation. Previous studies suggest that Nrf2 may be constitutively activated in NSCLC. Based on the above information, we investigated whether Prx1 and/or Nrf2 levels have prognostic significance in stage I NSCLC. METHODS AND RESULTS: Immunohistochemical expression of Prx1 and Nrf2 was evaluated in paraffin-embedded tissues from 90 patients who underwent a curative surgical resection. Increased expression of cytosolic Prx1 (66.7%) and nuclear Nrf2 (61.8%) was observed in this series. Prx1 elevation, but not Nrf2, correlated with reduced recurrence-free survival and overall survival on univariate (P = 0.01 and P = 0.03) and multivariate (P = 0.003 and P = 0.005) analyses. CONCLUSION: This is the first study to test the prognostic significance of Prx1 and Nrf2 in human cancers. Our results show that Prx1 expression status predicts for recurrence and shorter survival in stage I NSCLC after surgery. Considering the possible role of Prx1 and Nrf2 in radioresistance/chemoresistance, it warrants future investigation to evaluate whether elevated Prx1 and/or Nrf2 levels are predictive of treatment response in advanced lung cancer and other malignancies.

High superoxide dismutase and low glutathione peroxidase activities in red blood cells predict susceptibility of lung cancer patients to radiation pneumonitis.

Radiation pneumonitis is an unpredictable complication of radiotherapy for lung cancer and a condition which can cause significant morbidity. The ability to identify patients at a high risk of developing pneumonitis is critical, since it will enable the individualization of the treatment plan. Because the cytotoxic effect of radiation is propagated through reactive oxygen species (ROS) and ROS-driven oxidative stress, the role of antioxidant defense systems in radiation pneumonitis was investigated. Using the pneumonitis-sensitive C3H/HeN mice as a model, we demonstrated that the antioxidant response of the lung correlated well with that of red blood cells (RBC). We then proceeded to test whether differences of RBC antioxidant response would predict the pneumonitis development in patients. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities and glutathione in RBC were measured at baseline and then weekly for 6 weeks of treatment in 15 eligible patients receiving concurrent chemo-radiotherapy for unresectable stage III NSCLC. Striking differences were found in the antioxidant activities of RBC with respect to the pneumonitis development. Those who developed pneumonitis showed higher SOD and lower GPX activities at baseline compared to those who did not (3.7 vs 6.8 units/mg for median SOD, 16.5 vs 10.7 nmol/min/mg for median GPX). The functional imbalance of SOD and GPX was displayed consistently throughout the treatment period. The sensitivity and specificity of pneumonitis prediction were further increased when the GPX/SOD ratio was analyzed (pretreatment P = 0.0046). Our results provide a strong rationale to monitor SOD and GPX activities of RBC to identify patients who are at risk of developing pneumonitis, and to implement a strategy of increasing the GPX/SOD ratio in order to lower the risk.