Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia.

Previously, we found that dysfunctional natural killer (NK) cells with low interferon gamma (IFN-γ) were restored in acute myeloid leukemia (AML) by the FLT4 antagonist MAZ51. Here, we developed 12 peptides targeting FLT4 for clinical application and examined whether they restored the frequency of lymphocytes, especially T cells and NK cells, and high IFN-γ expression, as MAZ51 treatment did in our previous study. Although clinical data from using peptides are currently available, peptides targeting FLT4 to modulate immune cells have not been fully elucidated. In this study, we focus on novel peptide 4 (P4) from the intracellular domain of FLT4 because it had dominant negative activity. Similar to MAZ51, high IFN-γ levels were expressed in AML-mononuclear cells exposed to P4. Additionally, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was treated. Interestingly, the regulatory T cells were significantly decreased by P4, implying the role of peptide in tumor niche. Overall, we demonstrated the therapeutic value of functionally modulating lymphocytes using a peptide targeting FLT4 and proposed the development of advanced therapeutic approaches against AML by using immune cells.

Peptides Targeting Fms-Related Tyrosine Kinase-4 Activate the Function of Natural Killer Cells in Acute Myeloid Leukemia.

BACKGROUND AND OBJECTIVES: The increased expression for the Fms-related tyrosine kinase-4 (FLT-4, known as VEGFR-3) is relevant to dysfunctional natural killer (NK) cells in acute myeloid leukemia (AML). MAZ51 (M), a VEGFR-3 inhibiting chemical, was effectively restored the function of NK cells via the high expression of interferon- gamma (IFN-γ) in NK cells, as shown in our previous study. Although tremendous amount of clinical data using peptides are currently available in real clinic, peptides targeting FLT-4 in modulating immune cells such as NK cells are not fully elucidated. METHODS AND RESULTS: In present study, we developed peptides targeting FLT-4 (P), which is inhibiting an affinity for AML-NK expressing FLT-4 in vitro and in vivo. Bone marrow (BM) and peripheral blood (PB) mononuclear cells (MNCs) from AML patients were treated with combinational cocktails of the three agents including P, M, ara-C (A) and FLT-4 expression and IFN-γ release were examined. In an AML mouse model, IFN-γ expression were examined in T and NK cells from mouse BM, spleen, and liver to address relevance between peptides and immune cell activation. We found that AML-NK cells both in human and mouse samples showed a gradual increase the IFN-γ levels compared to the controls. There was a trend toward a reduction in leukemic blasts in the BM, spleen, and liver from the AML mice, when we compared the effects of combinational treatments. CONCLUSIONS: Our results suggest that the function of AML-NK cells was synergistically activated by P in combination with M or A.

Enhanced Anti-Leukemic Effects through Induction of Immunomodulating Microenvironment by Blocking CXCR4 and PD-L1 in an AML Mouse Model.

Previously, we found that dual therapy by the CXCR4 inhibitor Plerixafor and cytosine arabinoside (Ara-C) effectively eradicated leukemia cells and concurrently activated immune cells in acute myeloid leukemia (AML). To reveal the significance of programmed death-ligand1 (PD-L1) in AML and as a strategic approach, we investigated the anti-leukemic effect of a triple combinational therapy by utilizing Plerixafor and anti-PD-L1 in combination with chemotherapy in an AML mouse model. We examined leukemic myeloid blast cells in multiple organs after the successive treatment with Ara-C, Plerixafor, and anti-PD-L1. The results showed that noticeable benefits of triple combinational therapy for eradication of myeloid blast cells in vivo with prolonged survival rates. The frequencies of regulatory T cells (Tregs), monocytic-myeloid-derived suppressor cells (M-MDSCs), and granulocytic-myeloid-derived suppressor cells (G-MDSCs), in the peripheral blood of leukemic mice were consistently decreased, even when mice were sacrificed alive at D + 26 after completion of the triple combinational therapy, compared to the other subgroups. These findings imply that the modulation by the triple combinational therapy may lead to more efficient leukemic myeloid blast cell ablation through the suppression of Tregs or M-MDSCs and G-MDSCs in AML. Although Plerixafor and PD-L1 antagonist do not have a direct anti-leukemic role, our results provide some clues and guidelines to develop clinically therapeutic strategies for chemotherapy-resistant patients by the modulation of leukemic microenvironments.

Role of CXCR4 Antagonist in Megakaryocyte Reinstatement with Increased Sinusoidal Vessel Density.

The bone marrow microenvironment (BMM) provides a protective niche that supports the growth and survival of leukemic stem cells. It is known that a regulation of homing to BM and retention of hematopoietic stem cells (HSCs) occur by SDF-1/CXCR4 axis in BMM. Previously, we found that altering the BMM by the CXCR4 antagonist led to enhanced cytotoxic activity of immune cells, which leads to increased susceptibility of leukemic cells to chemotherapeutic agents such as cytosine arabinoside (Ara-C) in leukemic BMM. However, no reports have yet shown an architectural change of BMM such as the sinusoidal vessel and megakaryocyte by plerixafor treatment. Thus, we performed immunohistochemistry and observed that the capillary density of sinusoidal vessels was highly increased by CXCR4 antagonist with Ara-C in leukemia, showing the reconstruction of BMM with megakaryocytes in sinusoidal vessels by dual treatment. The number of megakaryocytes was also increased in the Plerixafor treated group, compared to that of leukemic or wild groups. Ultimately, we addressed the normalization of megakaryocyte and BMM in leukemia by showing the reconstitution of the sinusoidal vasculature by Plerixafor. This study proposed that chemotherapy with CXCR4 antagonist represents an advanced therapeutic strategy of targeting the leukemic niche.

Effect of High VEGF-C mRNA Expression on Achievement of Complete Remission in Adult Acute Myeloid Leukemia.

Although vascular endothelial growth factor-C (VEGF-C) is known to be expressed in acute myeloid leukemia (AML) blasts, the relevance of VEGF-C in the clinical setting remains to be fully explored. We examined the effect of VEGF-C on achievement of complete remission (CR) in adult de novo AML and immune cell population profiles according to VEGF-C mRNA expression. In comparison of VEGF-C expression between the no-CR and CR groups, the CR group showed a trend toward higher levels of plasma VEGF-C (P = .088), whereas mRNA expression of VEGF-C was downregulated (P = .008). Next, patients with continuous data for VEGF-C were divided into two groups (low vs. high) by a ROC curve analysis. The low- versus high-level groups for plasma VEGF-C (RR of 0.20, P = .030), mRNA expression of VEGF-C (RR of 18.75, P = .003), and the ratio of plasma level to mRNA expression (RR of 0.05, P = .007) were potential predictors of CR on univariate analysis. After adjusting for potential clinical factors including genetic group, multivariate analyses revealed that high VEGF-C mRNA expression was an independent risk factor for failure of induction chemotherapy. Furthermore, patients with high VEGF-C mRNA expression had a lower frequency of NKT and CD8+ cells and showed a trend for a lower frequency of NK cells. These results suggest that interruption of VEGF-C signaling might be a potential therapeutic target for antileukemic treatment in AML patients.

Antitumor activity of water extract of a mushroom, Inonotus obliquus, against HT-29 human colon cancer cells.

In the current study, it was demonstrated that the hot water extract of I. obliquus (IOWE) exerts inhibitory activity against the proliferation of human colon cancer cells (HT-29). The inhibitory effect of IOWE on the growth of HT-29 cancer cells was evaluated by treating cells with IOWE at concentrations of 0.25, 0.5 and 1.0 mg/mL for 24 or 48 h. The IOWE inhibited cell growth in a dose-dependent manner, and this inhibition was accompanied by apoptotic cell death. The maximum inhibitory effect (56%) was observed when IOWE was treated at a concentration of 1.0 mg/mL for 48 h. The apoptotic effect of IOWE on HT-29 cells was also confirmed by flow cytometric analysis. In addition, the apoptotic cell percentage was closely associated with down-regulation of Bcl-2 and up-regulation of Bax and caspase-3. The results suggest that IOWE would be useful as an antitumor agent via the induction of apoptosis and inhibition of the growth of cancer cells through up-regulation of the expression of proapoptotic proteins and down-regulation of antiapoptotic proteins.

Anti-obesity and antidiabetic effects of deep sea water on ob/ob mice.

Recently, deep sea water (DSW) has started to receive much attention for therapeutic intervention in some lifestyle diseases. In this study, the anti-obesity and antidiabetic effects of DSW in ob/ob mice were investigated. The animals were randomly divided into two groups with six animals: control group received tap water; the experimental group was treated with DSW of hardness 1000 for 84 days. The body weight gain after 84 days in DSW-fed group was decreased by 7% compared to the control group. The plasma glucose levels in the DSW-fed mice were substantially reduced by 35.4%, as compared to control mice. The results of oral glucose tolerance test revealed that DSW-fed groups significantly increased the glucose disposal after 84 days. DSW increased plasma protein levels of adiponectin and decreased plasma protein levels of resistin, RBP4, and fatty acid binding protein. Moreover, GLUT4 and AMP-activated protein kinase levels in skeletal muscle tissue were increased while peroxisome proliferator-activated receptor gamma and adiponectin were decreased in adipose tissue of DSW-fed mice. These results suggest that the antidiabetic and anti-obesity activities of DSW were mediated by modulating the expression of diabetes- and obesity-specific molecules. Taken together, these results provide a possibility that continuous intake of DSW can ameliorate obesity and diabetes.

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells.

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.

Apoptosis of human hepatocarcinoma (HepG2) and neuroblastoma (SKN-SH) cells induced by polysaccharides-peptide complexes produced by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala.

Three different polysaccharide-peptide complexes (PPC, named as Fr-I, Fr-II, and Fr-III) were produced by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala, and their anticancer activities were investigated in human hepatocarcinoma (HepG2) and neuroblastoma (SK-N-SH) cells. The highest inhibitory effects of PPC on both HepG2 and SK-N-SH cells were achieved with Fr-I, whereas Fr-III with low molecular mass showed lower inhibition effects. Interestingly, the inhibitory effects of the three fractions were increased after protease digestion, suggesting that the inhibitory effects resulted mainly from the carbohydrate moiety, at least in the case of Fr-II and Fr-III, of PPC. The results of DNA fragmentation in PPC-induced apoptotic cells were confirmed by both DNA ladder assay and comet assay. Our investigation also showed that PPC-induced apoptosis of both cancer cells was associated with intracellular events including DNA fragmentation, activation of caspase-3, and modulation of Bcl-2 and Bax. We conclude that PPC has potential as a novel therapeutic agent for the treatment of both HepG2 and SK-N-SH cancer cells without any cytotoxicity against normal cells.

Production of extracellular polysaccharides by submerged mycelial culture of Laetiporus sulphureus var. miniatus and their insulinotropic properties.

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l(-1)) and EPS production (0.6 g l(-1)) in submerged flask culture were achieved in a medium containing 30 g l(-1) maltose, 2 g l(-1) soy peptone, and 2 mM MnSO(4).5H2O at an initial pH 2.0 and temperature 25 degrees C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l(-1), respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.