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.

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.

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.

Plasma proteome analysis for anti-obesity and anti-diabetic potentials of chitosan oligosaccharides in ob/ob mice.

Altered levels of adipokines, derived as a result of distorted adipocytes, are the major factors responsible for changing biochemical parameters in obesity that leads to the development of metabolic disorders such as insulin resistance and atherosclerosis. In our previous reports, chitosan oligosaccharides (CO) were proved to inhibit the differentiation of 3T3-L1 adipocytes. In the present study, an attempt was made to investigate the anti-obesity and anti-diabetic effect of CO on ob/ob mice, by means of differential proteomic analysis of plasma. This was followed by immunoblotting, and gene expression in adipose tissue to clarify the molecular mechanism. CO treatment showed reduced diet intake (13%), body weight gain (12%), lipid (29%) and glucose levels (35%). 2-DE results showed differential levels of five proteins namely RBP4, apoE, and apoA-IV by >2-fold down-regulation and by >2-fold of apoA-I and glutathione peroxidase (GPx) up-regulation after CO treatment. Immunoblotting studies of adiponectin and resistin showed amelioration in their levels in plasma. Furthermore, the results of gene expressions for adipose tissue specific TNF-alpha, and IL-6 secretary molecules were also down-regulated by CO treatment. Gene expressions of PPAR gamma in adipose tissue were in good agreement with the ameliorated levels of adipokines, thereby improving the pathological state. Taken together, CO might act as a potent down-regulator of obesity-related gene expression in ob/ob mice that may normalize altered plasma proteins to overcome metabolic disorders of obesity.

Inhibitory effect of deep-sea water on differentiation of 3T3-L1 adipocytes.

Currently, the utilization of deep-sea water (DSW) is receiving much attention due to its high productivity, large quantity, and potential for biological application. The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days, confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. In this study, the effects of DSW on differentiation adipocyte 3T3-L1 cells were studied. DSW significantly decreased lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. DSW of hardness 1,000 was the most effective for inhibiting adipocyte differentiation without any cytotoxicity. DSW significantly reduced expression mRNA levels of PPARgamma and C/EBPalpha and protein levels of fatty-acid-binding protein and adiponectin. Our results suggest a potential role for DSW as anti-obesity agents by inhibiting adipocyte differentiation mediated through the down-regulated expression of adipogenic transcription factors and adipocyte-specific proteins.

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.

Proteomic analysis in ob/ob mice before and after hypoglycemic polysaccharide treatments.

In an attempt to discover novel biomarker proteins in type 2 diabetes prognosis, we investigated the influence of hypoglycemic extracellular polysaccharides (EPS) obtained from the macrofungus Tremella fuciformis on the differential levels of plasma proteins in ob/ob mice using two-dimensional gel electrophoresis (2-DE). The 2-DE analysis demonstrated that 92 spots from about 900 visualized spots were differentially regulated, of which 40 spots were identified as principal diabetes-associated proteins. By comparing control with EPS-fed mice, we found that at least six proteins were significantly altered in ob/ob mice, including Apo A-I, IV, C-III, E, retinol-binding protein 4, and transferrin, and their levels were interestingly normalized after EPS treatment. Western blot analysis revealed that the altered levels of the two regulatory molecules highlighted in diabetes and obesity (e.g., resistin and adiponectin) were also normalized in response to EPS. The Mouse Diabetes PCR Array profiles showed that the expression of 84 genes related to the onset, development, and progression of diabetes were significantly downregulated in liver, adipocyte, and muscle of ob/ob mice. EPS might act as a potent regulator of gene expression for a wide variety of genes in ob/ob mice, particularly in obesity, insulin resistance, and complications from diabetes mellitus.

Proteomic and transcriptomic analysis for streptozotocin-induced diabetic rat pancreas in response to fungal polysaccharide treatments.

In an attempt to search for novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (EPS) on the differential change in pancreatic proteome and transcriptome in streptozotocin (STZ)-induced diabetic rats using 2-DE-based protein mapping and oligonucleotide microarray analysis. The 2-DE system separated more than 2000 individual spots, demonstrating that 34 proteins out of about 500 matched spots were differentially expressed. A total of 22 overexpressed and 12 underexpressed proteins in 2-DE map were observed (p<0.05) between the healthy and diabetic rats, of which 26 spots were identified by PMF analysis. Of these, significant down regulation of carbonyl reductase (Cbr), hydroxymethylglutaryl-CoA synthase (HMGCS), and putative human mitogen-activated protein kinase activator with WD repeats-binding protein (MAWDBP) in diabetic pancreas were reported for the first time in this study. When treated with EPS, all these four proteins were reverted to normal levels. The microarray analysis revealed that 96 out of 1272 genes were down- or up-regulated in the diabetic rats and the altered transcript levels of many of these genes were reversed after EPS treatment. In particular, ROS generation in rat islets was significantly increased after STZ treatment, thereafter EPS treatment was likely to play a preventive role in beta-cell destruction mediated by STZ. Taken together, EPS may act as a potent regulator of gene expression for a wide variety of genes in diabetic rats, particularly in antioxidative stress, insulin biosynthesis, and cell proliferation.

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.