Synthesis, structure and midkine binding of chondroitin sulfate oligosaccharide analogues.

The preparation of chondroitin sulfate (CS) oligosaccharide mimetics, more easily synthesized than natural sequences, is a highly interesting task because these compounds pave the way for modulation of the biological processes in which CS is involved. Herein, we report the synthesis of CS type E analogues which present easily accessible glucose units instead of glucuronic acid (GlcA) moieties. NMR experiments and molecular dynamics simulations showed that the 3D structure of these compounds is similar to the structure of the natural CS-E oligosaccharides. In addition, fluorescence polarization (FP) and saturation transfer difference NMR (STD-NMR) experiments revealed that the synthesized CS-like derivatives were able to interact with midkine, a model heparin-binding growth factor, suggesting that the presence of the GlcA carboxylate groups is not essential for the binding. Overall, our results indicate that the synthesized glucose-containing oligosaccharides can be considered as functional and structural CS mimetics.

Shedding more light on the role of Midkine in hepatocellular carcinoma: New perspectives on diagnosis and therapy.

One of the most common malignant tumors is hepatocellular carcinoma (HCC). Progression of HCC mainly results from highly complex molecular and pathological pathways. Midkine (MDK) is a growth factor that impacts viability, migration, and other cell activities. Since MDK has been involved in the inflammatory responses, it has been claimed that MDK has a crucial role in HCC. MDK acts as an anti-apoptotic factor, which mediates tumor cell viability. In addition, MDK blocks anoikis to promote metastasis. There is also evidence that MDK is involved in angiogenesis. It has been shown that the application of anti-MDK approaches might be promising in the treatment of HCC. Besides, due to the elevated expression in HCC, MDK has been proposed as a biomarker in the prognosis and diagnosis of HCC. In this review, we will discuss the role of MDK in HCC. It is hoped that the development of new strategies concerning MDK-based therapies will be promising in HCC management.

Pharmacokinetics of midkine with different N-terminal structures in rats.

Midkine (MK) is a heparin-binding growth factor that functions in multiple physiological processes, making it a promising drug target for treating various diseases including osteoarthritis (OA). However, the lack of pharmacokinetic studies on MK limits further clinical research. As the N-domain of MK protein appears to be more important for its stability, this study aimed to investigate the pharmacokinetic profiles of recombinant human (rh)MK with different structures at the N-terminus via different administration routes in rats and guinea pigs. A single intramuscular (IM) injection of 1 mg/kg rhMK with or without extended sequences at the N-terminus expressed by E. coli or Pichia was administered to six male SD rats. rhMK concentrations in sequential tail blood samples were measured by ELISA. rhMK without extended N-terminal sequences expressed by Pichia had a greater area under the curve (AUC), slower clearance, and longer half-life in rats following a single IM injection than those of the other rhMK proteins. The AUC values for rhMK after IM and intra-articular (IA) administration were 1523.3 ± 35.2 h × ng/mL and 872.0 ± 36.1 h × ng/mL, whereas the apparent volumes of distribution (Vd/f) were 0.184 ± 0.067 L/kg and 11.6 ± 0.8 L/kg, respectively, suggesting that rhMK was distributed more locally after IA injection than after IM injection as Vd/f magnitude gives a general idea of extent distribution in the body and higher Vd/f represents more locally distribution. rhMK concentration in the articular cartilage was markedly higher than that in serum and reached the highest level at 3 days after a single IA injection in Hartley guinea pigs. As the dose increased from 10 to 50 mg/kg, the AUC increased in a greater-than-dose-proportional manner, suggesting that rhMK exhibits non-linear pharmacokinetics in rats after a single IM injection in this dose range. These results indicated that the N-terminal structure and administration route have substantial effects on the pharmacokinetics of rhMK in rats. Furthermore, rhMK was maintained in articular cartilage with minimal diffusion into the blood following IA injection in Hartley guinea pigs, providing a foundation for clinical research on the use of rhMK for OA treatment via IA delivery.

The Possible Interactions and Therapeutic Roles of Lithium Chloride and Midkine on Cancer Treatment.

Midkine (MK) is a heparin-binding anti-apoptotic growth factor or cytokine also known as neurite growth-promoting factor 2 (NEGF2). It is developmentally an important retinoic acid-responsive gene product strongly induced during the mid-gestation stage. Midkine promotes different cellular events such as cell growth, differentiation, survival, gene expression, and drug resistence. Midkine, the phosphatidylinositol 3-kinases (PI3-kinase, PI3K) and glycogen synthase kinase-3 beta (GSK-3ß) inhibitors together with lithium chloride may be a very effective treatment modality, especially in tumors with high expression of these two molecules. PI3 kinase and GSK-3ß, both serine threonine kinases located in the center of the signaling network, are very important regulator molecules for cell survival or death. Lithium chloride (LiCl), with its newly discovered antineoplastic effect and cytotoxicity potentiation, has become a promising agent in the application of new combination treatments. Although the LiCl mechanism of action is still not fully understood, previous studies have shown that LiCl is an inhibitör of the inositol monophosphatase (IMPase) and GSK-3ß. GSK-3ß, is a serine-threonine protein kinase involved in cell proliferation, differentiation, survival, apotosis, and tumorogenesis. The role of GSK-3ß in tumorigenesis and cancer remains controversial. It may have a function as a tumor suppressor for certain types of tumors, but it promotes cell growth and development in other tumor types.

Synthesis of a Fluorous-Tagged Hexasaccharide and Interaction with Growth Factors Using Sugar-Coated Microplates.

Here, we report the synthesis of a sulfated, fully protected hexasaccharide as a glycosaminoglycan mimetic and the study of its interactions with different growth factors: midkine, basic fibroblast growth factor (FGF-2) and nerve growth factor (NGF). Following a fluorous-assisted approach, monosaccharide building blocks were successfully assembled and the target oligosaccharide was prepared in excellent yield. The use of more acid stable 4,6-O-silylidene protected glucosamine units was crucial for the efficiency of this strategy because harsh reaction conditions were needed in the glycosylations to avoid the formation of orthoester side products. Fluorescence polarization experiments demonstrated the strong interactions between the synthesized hexamer, and midkine and FGF-2. In addition, we have developed an alternative assay to analyse these molecular recognition events. The prepared oligosaccharide was non-covalently attached to a fluorous-functionalized microplate and the direct binding of the protein to the sugar-immobilized surface was measured, affording the corresponding KD,surf value.

Identification and functional characterization of amphioxus Miple, ancestral type of vertebrate midkine/pleiotrophin homologues.

Midkine (MK) and pleiotrophin (PTN) are the only two members of heparin-binding growth factor family. MK/PTN homologues found from Drosophila to humans are shown to have antibacterial activities and their antibacterial domains are conserved during evolution. However, little is known about MK/PTN homologue in the basal chordate amphioxus, and overall, information regarding MK/PTN homologues is rather limited in invertebrates. In this study, we identified a single MK/PTN homologue in Branchiostoma japonicum, termed BjMiple, which has a novel domain structure of PTN-PTNr1-PTNr2, and represents the ancestral form of vertebrate MK/PTN family proteins. BjMiple was expressed mainly in the ovary in a tissue-dependent fashion, and its expression was remarkably up-regulated following challenge with bacteria or their signature molecules LPS and LTA, suggesting its involvement in antibacterial responses. Functional assays revealed that BjMiple had strong antimicrobial activity, capable of killing a panel of Gram-negative and Gram-positive bacteria via a membranolytic mechanism, including interaction with bacterial membrane via LPS and LTA, membrane depolarization and high intracellular levels of ROS. Importantly, strong antibacterial activity was localized in PTN42-61 and PTNr142-66. Additionally, BjMiple and its derived peptides PTN42-61 and PTNr142-66 were not cytotoxic to human RBCs and mammalian cells. Taken together, our study suggests that amphioxus Miple is the ancestral type of vertebrate MK/PTN family homologues, and can play important roles as innate peptide antibiotics, which renders it a promising template for the design of novel peptide antibiotics against multi-drug resistant bacteria.