Isolation of MLL1 Inhibitory RNA Aptamers

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers—APT1 and APT2—represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5′-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3′. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

Methanolic Extract Isolated from Root of Lycoris aurea Inhibits Cancer Cell Growth and Endothelial Cell Tube Formation In Vitro

In this study, we investigated the effect of methanolic extract isolated from the root of Lycoris aurea (LA) on the growth of cancer cells and the tube formation activity of endothelial cells. Various cancer cells were treated with LA at doses of 0.3, 1, 3, 10 or 30 microg/ml and LA significantly suppressed the growth of several cancer cell lines, including ACHN, HCT-15, K-562, MCF-7, PC-3 and SK-OV-3, in a dose-dependent manner. We also found that LA induced cell cycle arrest at G2/M phase in ACHN renal cell adenocarcinoma cells. Further study demonstrated that LA concentration-dependently inhibited the tube formation, which is a widely used in vitro model of reorganization stage of angiogenesis, in human umbilical vein endothelial cells. Collectively, these results show that LA inhibits the growth of cancer cells and tube formation of endothelial cells and the growth-inhibitory effect of LA might be mediated, at least in part, by blocking cell cycle progression.

Dendritic Cell Activation by Glucan Isolated from Umbilicaria Esculenta

BACKGROUND: Lichen-derived glucans have been known to stimulate the functions of immune cells. However, immunostimulatory activity of glucan obtained from edible lichen, Umbilicaria esculenta, has not been reported. Thus we evaluated the phenotype and functional maturation of dendritic cells (DCs) following treatment of extracted glucan (PUE). METHODS: The phenotypic and functional maturation of PUE-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. PUE-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity. Finally we detected the activation of MAPK and NF-kappaB by immunoblot. RESULTS: Phenotypic maturation of DCs was shown by the elevated expressions of CD40, CD80, CD86, and MHC class I/II molecules. Functional activation of DCs was proved by increased cytokine production of IL-12, IL-1beta, TNF-alpha, and IFN-alpha/beta, decreased endocytosis, and enhanced proliferation of allogenic T cells. Polymyxin B, specific inhibitor of lipopolysaccharide (LPS), did not affect PUE activity, which suggested that PUE was free of LPS contamination. As a mechanism of action, PUE increased phosphorylation of ERK, JNK, and p38 MAPKs, and enhanced nuclear translocation of NF-kappaB p50/p65 in DCs. CONCLUSION: These results indicate that PUE induced DC maturation via MAPK and NF-kappaB signaling pathways.

Histone deacetylase inhibitor KBH-A42 inhibits cytokine production in RAW 264.7 macrophage cells and in vivo endotoxemia model

In light of the anti-inflammatory properties of histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), we examined a new HDAC inhibitor KBH-A42 for its anti-inflammatory activities. KBH-A42 showed noteworthy anti-inflammatory properties in vitro via suppression of the production of TNF-alpha, a proinflammatory cytokine, and nitric oxide (NO), a proinflammatory effector molecule, in LPS-stimulated RAW264.7 cells and peritoneal macrophages. It also inhibited TNF-alpha production in vivo as demonstrated in a LPS-induced mouse endotoxemia model. The levels of TNF-alpha, IL-1beta, IL-6 and iNOS mRNAs determined by RT-PCR propose that the inhibition of these pro-inflammatory mediators by KBH-A42 resulted from inhibiting expression of these genes. However, the EMSA study to see the effect of KBH-A42 on the binding of NF-kappaB, a transcription factor, to a specific DNA sequence showed that the binding of NF-kappaB to DNA was not changed regardless of increasing the concentration of KBH-A42 in the presence and absence of LPS stimulation. Interestingly, DNA binding of another transcription factor AP-1 dose-dependently increased by KBH-A42. KBH-A42 differentially regulated the phosphorylation of MAP kinases. While the phosphprylation of ERK1/2 and SAPK/JNK was not affected by KBH-A42, the phosphorylation of p38 decreased by KBH-A42. These results showed that KBH-A42 inhibits production of proinflammatory cytokines in macrophages by decreasing their mRNA levels, and p38 kinase is involved in the KBH-A42-mediated inhibition.

Effect Of Serum Isolated From Osteoporotic Human On The Growth And Differentiation Of Normal Human Osteoblast

Prevention of reperfusion injury with CD18 monoclonal antibody and superoxide dismutase

Prolonged ischemia results in cellular necrosis and only prompt restoration of blood flow will prevent this type of injury. However, reperfusion itself can cause significant injury of previously ischemic tissue, i.e. "reperfusion injury'. This is an issue of concern in many areas of reconstructive surgery including free tissue transfer and replantation. Many factors have been implicated in the cause of reperfusion injury. Oxygen free radicals have enjoyed increasing popularity recently, but leukocytes had been thought to have a role only in the healing process that follows ischemic injury. Current studies in myocardium, liver and intestine have shown a dramatic increase in tissue leukocytes after ischemia-reperfusion and evidence implicating leukocytes in pathogenesis of ischemia-reperfusion injury has come from studies demonstrating significant injury reduction by depletion of circulating neutrophils. Therefore, increased neutrophil adhesiveness is a critical early step in the sequence of events leading to neutrophil-mediated injury. The purpose of this study is to evaluate the effect of CDl8 monoclonal antibody(CDl8 mAb), blocking antibody of neutrophil adherence, and superoxide dismutase (SOD), free radical scavenger, on reperfusion injury in rat epigastric island skin flap. The epigastric pedicle was occluded for six hours with ambient temperature at 22+/-1degrees C. The epigastric nerve was carefully dissected out and left intact to minimize autocannibalization. The flaps were sutured back down to their beds over interposed silicone sheets to prevent plasmatic imbibition. Fifteen minutes before reperfusion, the flaps were perfused with saline, CDl8 mAb(1 mg/kg), SOD(20,000 unit/kg) or CDl8 mAh/SOD(1 mg/kg + 20,000unit/kg). Percentage of flap survival was assessed by computerized planimetry on the seventh day. Tissue biopsies for myeloperoxidase(MPO) and malonyldialdehyde (MDA) were obtained at 24 hours after reperfusion. The results were as follows. 1. Percentage of flap survival was significantly increased in CDl8 mAb/SOD, CDl8 mAb and SOD groups in order, compared to the control(P < 0.05). Percentage of flap survival was significantly increased in CDl8 mAb group as compared with SOD group(p < 0.05). Percentage of flap survival significantly increased in CDl8 mAb/SOD group as compared with CDl8 mAb and SOD groups(p < 0.05) 2. MPO activity was significantly decreased in CDl8 mAb/SOD, CDl8 mAb and SOD groups(p < 0.01). MPO activity was significantly decreased in CDl8 mAb group as compared with SOD group. (p < 0.01). 3. MDA content was significantly decreased in CDl8 mAb/SOD, CDl8 mAb and SOD groups (p < 0.01), but the difference between CDl8 mAb and SOD groups was not significant. From those above results, we get to the conclusion that blocking neutrophil adherence and/or aggregation with monoclonal antibodies to CDl8 as compared with radical scavenger significantly ameliorates reperfusion injury. It is suggested that combination of modalities with antiadhesion therapy and radical scavenger may have a synergistic effect of improving flap survival and may be the optimal prevention of ischemiareperfusion injury.

Prevention of reperfusion injury with CD18 monoclonal antibody and superoxide dismutase

Prolonged ischemia results in cellular necrosis and only prompt restoration of blood flow will prevent this type of injury. However, reperfusion itself can cause significant injury of previously ischemic tissue, i.e. "reperfusion injury'. This is an issue of concern in many areas of reconstructive surgery including free tissue transfer and replantation. Many factors have been implicated in the cause of reperfusion injury. Oxygen free radicals have enjoyed increasing popularity recently, but leukocytes had been thought to have a role only in the healing process that follows ischemic injury. Current studies in myocardium, liver and intestine have shown a dramatic increase in tissue leukocytes after ischemia-reperfusion and evidence implicating leukocytes in pathogenesis of ischemia-reperfusion injury has come from studies demonstrating significant injury reduction by depletion of circulating neutrophils. Therefore, increased neutrophil adhesiveness is a critical early step in the sequence of events leading to neutrophil-mediated injury. The purpose of this study is to evaluate the effect of CDl8 monoclonal antibody(CDl8 mAb), blocking antibody of neutrophil adherence, and superoxide dismutase (SOD), free radical scavenger, on reperfusion injury in rat epigastric island skin flap. The epigastric pedicle was occluded for six hours with ambient temperature at 22+/-1degrees C. The epigastric nerve was carefully dissected out and left intact to minimize autocannibalization. The flaps were sutured back down to their beds over interposed silicone sheets to prevent plasmatic imbibition. Fifteen minutes before reperfusion, the flaps were perfused with saline, CDl8 mAb(1 mg/kg), SOD(20,000 unit/kg) or CDl8 mAh/SOD(1 mg/kg + 20,000unit/kg). Percentage of flap survival was assessed by computerized planimetry on the seventh day. Tissue biopsies for myeloperoxidase(MPO) and malonyldialdehyde (MDA) were obtained at 24 hours after reperfusion. The results were as follows. 1. Percentage of flap survival was significantly increased in CDl8 mAb/SOD, CDl8 mAb and SOD groups in order, compared to the control(P < 0.05). Percentage of flap survival was significantly increased in CDl8 mAb group as compared with SOD group(p < 0.05). Percentage of flap survival significantly increased in CDl8 mAb/SOD group as compared with CDl8 mAb and SOD groups(p < 0.05) 2. MPO activity was significantly decreased in CDl8 mAb/SOD, CDl8 mAb and SOD groups(p < 0.01). MPO activity was significantly decreased in CDl8 mAb group as compared with SOD group. (p < 0.01). 3. MDA content was significantly decreased in CDl8 mAb/SOD, CDl8 mAb and SOD groups (p < 0.01), but the difference between CDl8 mAb and SOD groups was not significant. From those above results, we get to the conclusion that blocking neutrophil adherence and/or aggregation with monoclonal antibodies to CDl8 as compared with radical scavenger significantly ameliorates reperfusion injury. It is suggested that combination of modalities with antiadhesion therapy and radical scavenger may have a synergistic effect of improving flap survival and may be the optimal prevention of ischemiareperfusion injury.

Secondary behavior changes of fibroblast functions induced by conditioned medium of silicone-sensitized murine lymphocytes

Fibrous capsular contracture has been considered as a major side effect of breast silicone implant. The etiology of fibrous capsular contracture has not been fully determined. In the present study, we tried to determine the indirect effect of immune system in fibroblast function which plays a major role in fibrous capsular contracture. For preparation of conditional medium of lymphocytes, mouse (ICR) splenocytes were cultured for one day. Two kinds of conditioned medium, silicone conditioned medium (SCM) and silicone free normal conditioned medium (NCM), were prepared from splenocyte cell suspension cultured on silicone gel coated surface and naked surface, respectively. Mouse fibroblasts (NIH 3T3) were cultured in usual culture dish. Conditioned medium of 25% concentration was added. On day 2 after innoculation, cell number, thymidine incorporation and proline uptake of fibroblasts were measured. The results were as follow; 1) There was no difference of fibroblast number by cultivation in SCM of splenocytes compared with that in fresh medium(FM). 2)There was significant increase of DNA synthesis of fibroblasts in SCM compared with that in FM (p < 0.001). 3) There was significant increase of collagen synthesis of fibroblasts in SCM compared with that in FM (p < 0.01) and in NCM (p <0.001). 4) The functional activities of DNA and collagen synthesis mediated by same number of fibroblast were calculated to be significantly increased in SCM compared with that in NCM and in FM (p < 0.001). In conclusion, fibroblasts cultured in SCM had a higher potential to synthesize macromolecules such as collagen and DNA. We can postulate that SCM may contain certain amount of unknown growth stimulant of fibroblasts, which is produced by silicone gel sensitized murine splenocytes.

In vitro stimulation of nih 3t3 fibroblast proliferation by interleukin-6 and tumor necrosis factor-alpha generated from peritoneal macrophages activated by silicone gel

Silicone gel breast implants may induce local(fibrous capsular contracture) or systemic(rheumatoid arthritis, systemic sclerosis, etc) complications. The exact mechanism of fibrous capsular contracture has not been fully understood. In the present study, we tried to find out the effect of silicone gel on the fibroblast proliferation which has been known as a major contributing factor in fibrous capsular contracture formation. In vitro, activated macrophages are known to secrete monokines which affect fibroblast proliferation and collagen synthesis. And tumour necrosis factor-alpha(TNF-alpha) and interleukin-6(IL-6), which were released by macrophages, were reported as potent stimulator of fibroblast proliferation. The goal of this study is to investigate the role of macrophages and tumour necrosis factor-alphaor interleukin-6 in the interaction of fibroblasts and silicone gel. We designed four groups, two experimental and two control, using Institute for Cancer Research(ICR) mouse peritioneal macrophage and silicone gel. For the preparation of the conditioned medium of macrophages, peritoneal macrophages were prepared and cultured for 24 hours on the silicone gel-coated and naked (not coated) surface [silicone gel-macrophage conditioned medium(SCM; experimental group) and normal polystyrene-macrophage conditioned medium(NCM; control group) respectively]. To correct the effect of 10% fetal bovine serum which was included in Rapid Prototyping and Manufacturing Institute (RPMI) 1640 medium and draw the effect only by macrophages, the RPMI 1640 medium with 10% fetal bovine serum was cultured by the same method on the silicone gel-coated and naked surface (silicone gel-macrophage free conditioned medium; SFM and normal polystyrene-macrophage free conditioned medium; NFM respectively). Each conditioned medium was added onto NIH 3T3 fibroblasts culture at a final 25% concentration of total culture medium and followed by the cultivation for 24 hours. For antibody neutralizing experiments, each conditioned medium was preincubated with polyclonal rabbit anti-mouse TNF-alpha antibody or polyclonal rat anti-mouse IL-6 antibody for 1 hour and then, conditioned medium with antibody was added to the culture medium of NIH 3T3 fibroblasts by the same method. After 24 hours cultivation, total number of viable fibroblast(cell growth), DNA synthesis and collagen synthesis of fibroblasts with each medium were measured by sulforhodamine B(SRB) assay, 3H-thymidine and 3H-proline incorporation respectively. The results were as follows: 1. In the experiment about the effect of the conditioned medium on the fibroblast activity, the experimental group(SCM), compared with the control group(NCM), showed a significant increase of the cell growth (p<0.01), a significant decrease of DNA synthesis(p<0.001), but no significant difference in the collagen synthesis. 2. In the experiment about the effect of polyclonal rabbit anti-mouse TNF-alpha antibody on the fibroblast activity, after the addition of antibody the experimental group, compared with the control group, showed a significant decrease of the cell growth(p<0.001), a significant increase of DNA synthesis(p<0.01), but no significant difference in the collagen syn thesis. 3. In the experiment about the effect of polyclonal rat anti-mouse IL-6 antibody on the fibroblast activity, after the addition of antibody the experimental group, compared with the control group, showed a significant decrease of the cell growth(p<0.001), a significant increase of DNA synthesis(p<0.0001), but no significant difference in the collagen synthesis. In conclusion, culture supernatants (conditioned medium) of peritoneal macrophages, activated by silicone gel, stimulate the NIH 3T3 fibroblast proliferation. TNF-alpha and IL-6, products of macrophage, are involved in the stimulation of NIH 3T3 fibroblast proliferation in an in vitro condition.

Anti-tumor Effect of the Complex of Acriflavine and Guanosine (AG60) / 대한암학회지

PURPOSE: The anti-tumor effect of the complex of acriflavine and guanosine (AG60) was investigated. MATERIALS AND METHODS: In vitro cytotoxicity of AG60 was measured using SRB assay, and in vivo antitumor activity of AG60 was examined in CDF1 mice intraperitoneally inoculated with the P388 leukemic cells and in ICR mice inguinally implanted with S-180 cells. Tumor size and mean survival time were determined. RESULTS: AG60 and acriflavine showed strong anti-tumor effect in vitro on lung cancer (A549), renal cancer (UO-31) and colon cancer (COLO205) cells. However, AG60 did not show the cytotoxicity against normal cell line, 3T3. The range of the IC50 of AG60 to the various tumor cell lines was 0.09 microgram/ml through 1.94 microgram/ml. The treatment of ascitic tumor bearing CDF1 mice with AG60 resulted in over 160% increases in the mean survival time. The most effective dose of AG60 was 30 mg/kg body weight in tumor implanted mice. In solid tumor bearing ICR mice tumor growth and progression were suppressed in response to the different doses at 30 days; 69.8% suppression of tumor size in response to acriflavine, 16.0% to guanosine, 87.7% to AG60 and 78.5% to doxorubicin. In addition, 35% increases were observed in the means survival time of AG60 treated group compared with control group. CONCLUSION: The prominant anti-tumor effects of AG60 shown in this report would represent the possibility of the clinical trials.