Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Requirement of chondroitin sulfate/dermatan sulfate recognition in midkine-dependent migration of macrophages

Midkine (MK) is a heparin-binding growth factor that promotes cell migration, cell growth and cell survival. The promotion of migration of inflammatory cells, especially macrophages, by MK is involved in formation of a vascular abnormality, i.e. neointima formation. MK-induced migration of peritoneal exudate macrophages was inhibited by heparin, chondroitin sulfate E and dermatan sulfate, but not by chondroitin sulfate D or chondroitin 6-sulfate. Digestion of macrophages with chondroitinase ABC as well as chondroitinase B decreased the migratory activity. However, heparitinase digestion showed only slight effects. These results indicated that a chondroitin sulfate, i.e. an E-type oversulfated structure with dermatan sulfate domain, is involved in MK-induced migration of macrophages. Although a chondroitin sulfate proteoglycan, receptor-type protein tyrosine phosphatase (PTP ), participates in MK-induced migration of neurons and osteoblasts, PTP was not detected in macrophages. The MK-induced migration was inhibited by PP1, wortomanin, PD 98059 and vanadate, indicating that the downstream signaling system, which includes Src, PI3 kinase and ERK as important components, is shared with other MK signaling systems in which PTP is involved.     

siRNA targeting midkine inhibits gastric cancer cells growth and induces apoptosis involved caspase-3,8,9 activation and mitochondrial depolarization

Midkine (MK), a heparin-binding growth factor, is expressed highly in various malignant tumors, so it acts as attractive therapeutic target. In the present study, we used siRNA targeting MK to downregulate human MK expression in human gastric cancer cell line BGC823 and SGC7901 so as to determine the advantages of this anticancer therapeutic. The cell proliferation was evaluated by a WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate sodium salt) assay and colony formation assay. Apoptosis was determined by flow cytometer analysis and colorimetric assay. Our results showed that the BGC823 and SGC7901 cell growth were significantly inhibited by knockdown of MK gene. The loss of mitochondrial membrane potential, release of cytochrome c from the mitochondria into cytosol and increased activity of caspase-3, 8 and 9 occurred concomitantly with inhibition of MK gene. These results indicated that siRNA targeting MK gene can inhibit gastric cancer cells growth and induce apoptosis via mitochondrial depolarization and caspase-3 activation. MK siRNA may be a promising novel and potential therapeutic strategy for the treatment of gastric cancers.     

细胞因子人MK基因的克隆及在大肠杆菌中的高效表达(简报)

细胞因子Midkine(简称MK)是新发现的一类肝素结合因子家族中的一员。1988年,Kadamatsu等利用差异杂交法在经维甲酸诱导分化的小鼠畸胎瘤细胞株HM-1中首先克隆到小鼠MK基因。人MK基因则最早是从λgt10人胚肾(20-24周)cDNA库和EMBL-3人胎盘基因组库获得。成熟     

重组人中期因子midkine对大鼠膝关节软骨部分损伤的修复作用

目的:通过外源注射不同剂量的重组人中期因子midkine(rhMK),研究其对大鼠膝关节软骨部分损伤的修复作用。方法:雄性SD大鼠双侧膝关节建立软骨部分损伤的动物模型,术后24小时分别向关节腔内注射生理盐水或rhMK (20μg/kg、60μg/kg、180μg/kg)。于术后8周将大鼠全部处死,取材进行组织学观察,从而确定最佳注射剂量;在药代动力学研究中,按最佳注射剂量向正常大鼠膝关节腔内注射rhMK,分别于注射后1小时、1天、3天、6天、9天、12天和15天处死大鼠,检测膝关节软骨组织中rhMK的含量。结果:不同剂量的重组蛋白对膝关节软骨部分损伤均有不同程度的修复作用,其中180μg/kg的剂量效果最佳;以180μg/kg的剂量向正常大鼠膝关节腔内注射rhMK后,经过Kinetica5.0药代动力学软件拟合后,计算得rhMK在软骨组织中的消除相半衰期为8.69天。结论:rhMK对大鼠膝关节软骨部分损伤有明显的修复作用,最佳注射剂量为180μg/kg,最佳注射时间间隔为8天。     

Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage

Background

Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)β1-3N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood.

Methods

CS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration.

Results

The major disaccharide unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4-O-sulfate) (27.0%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy.

Conclusions and general significance

The NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.     

Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells

Effects of magnetic fields (MFs) on cancer cells may depend on cell type and exposure conditions. Gene expression levels are different among cancer cells. However, the effect of MFs on cancer cells with different gene expressions is still unclear. In this study, the cancer cell lines BGC-823, MKN-45, MKN-28, A549, SPC-A1, and LOVO were exposed to a low-frequency MF. Specific parameters of MFs were determined. Furthermore, the potential of the MF to influence cancer cell growth with midkine (MK) expression was evaluated. Cell proliferation and cell cycle were detected using the CCK-8 assay and flow cytometry. Cell ultrastructure was observed by transmission electron microscopy. BGC-823 cells with over-expression of MK (BGC-MK cells) and stanniocalcin-1 were generated by plasmid construction and transfection. Results showed that exposure to a 0.4-T, 7.5 Hz MF inhibited the proliferation of BGC-823, MKN-28, A549, and LOVO cells, but not MKN-45 and SPC-A1 cells. Moreover, the inhibitory effect of the MF on BGC-MK cells was lower (12.3%) than that of BGC-823 cells (20.3%). Analysis of the cell cycle showed that exposure to the MF led to a significant increase in the S phase in BGC-823 cells, but not in BGC-MK cells. In addition, organelle morphology was modified in BGC-823 cells exposed to the MF. These results suggest that exposure to a 0.4-T, 7.5 Hz MF could inhibit tumor cell proliferation and disturb the cell cycle. The alteration of MK expression in cancer cells may be related to the inhibitory effect of the MF on these cells.     

Overexpression of midkine contributes to anti-apoptotic effects in human meningiomas

Meningiomas are the second most common intracranial tumours. Most meningiomas grow slowly; however, atypical and anaplastic meningiomas show an aggressive biological behaviour. Overexpression of growth factors is considered to be a cause of carcinogenesis. Midkine and pleiotrophin are heparin-binding growth factors that promote growth, survival, migration and differentiation of various target cells. Both molecules are highly expressed during human embryogenesis but are rarely seen in the adult. We show that in relation to normal dura and arachnoid tissues, midkine was overexpressed in meningiomas on the mRNA and protein level, whereas pleiotrophin was not. Thereby, not only the intact but also the truncated form of midkine could be observed. The expression of midkine receptors was variable in different samples. Midkine stimulation of cultured meningioma cells induced phosphorylation of Akt, whereas no increase in phosphorylation of p42/44 MAPK or p38 MAPK could be detected. Midkine did not influence the proliferation of meningioma cells in vitro, but it did protect meningioma cells from camptothecin-mediated apoptotic cell death through reduction in the amounts of active caspase-3. These findings provide evidence for the overexpression of midkine in meningiomas which contributes to protection from cell death in these second most common intracranial tumours.     

Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.     

Pleiotrophin differentially regulates the rewarding and sedative effects of ethanol

Pleiotrophin (PTN) is a cytokine with important roles in dopaminergic neurons. We found that an acute ethanol (2.0 g/kg, i.p.) administration causes a significant up‐regulation of PTN mRNA and protein levels in the mouse prefrontal cortex, suggesting that endogenous PTN could modulate behavioural responses to ethanol. To test this hypothesis, we studied the behavioural effects of ethanol in PTN knockout (PTN^?/?) mice and in mice with cortex‐ and hippocampus‐specific transgenic PTN over‐expression (PTN‐Tg). Ethanol (1.0 and 2.0 g/kg) induced an enhanced conditioned place preference in PTN^?/? compared to wild type mice, suggesting that PTN prevents ethanol rewarding effects. Accordingly, the conditioning effects of ethanol were completely abolished in PTN‐Tg mice. The ataxic effects induced by ethanol (2.0 g/kg) were not affected by the genotype. However, the sedative effects of ethanol (3.6 g/kg) tested in a loss of righting reflex paradigm were significantly reduced in PTN‐Tg mice, suggesting that up‐regulation of PTN levels prevents the sedative effects of ethanol. These results indicate that PTN may be a novel genetic factor of importance in alcohol use disorders, and that potentiation of the PTN signalling pathway may be a promising therapeutic strategy in the treatment of these disorders.