Sadatoshi Sakuma

Serum midkine levels are increased in patients with various types of carcinomas

The level of expression of midkine (MK), a heparin-binding growth factor, is increased in many types of human carcinomas. An enzyme-linked immunoassay, which utilizes a combination of rabbit and chicken antibodies revealed that serum MK level in the controls (n= 135) was 0.154 ± 0.076 (mean ± SD) ng ml–1with an apparent cut-off value as 0.5 ng ml–1. Serum MK level was significantly elevated in the cancer patients (n= 150) (P< 0.001); 87% of the patients showed levels of more than 0.5 ng ml–1. All ten types of cancer examined showed a similar profile of serum MK level. There was no or weak correlation between C-reactive protein level, a marker of inflammation, and serum MK level. Furthermore, in case of gastric carcinoma and lung carcinoma, patients with stage I carcinoma already showed elevated serum MK levels. The present results indicated that serum MK could serve as a general tumour marker with a good potential for clinical application.

Neointima formation in a restenosis model is suppressed in midkine-deficient mice

Neointima formation is a common feature of atherosclerosis and restenosis after balloon angioplasty. To find a new target to suppress neointima formation, we investigated the possible role of midkine (MK), a heparin-binding growth factor with neurotrophic and chemotactic activities, in neointima formation. MK expression increased during neointima formation caused by intraluminal balloon injury of the rat carotid artery. Neointima formation in a restenosis model was strongly suppressed in MK-deficient mice. Continuous administration of MK protein to MK-deficient mice restored neointima formation. Leukocyte recruitment to the vascular walls after injury was markedly decreased in MK-deficient mice. Soluble MK as well as that bound to the substratum induced migration of macrophages in vitro. These results indicate that MK plays a critical role in neointima formation at least in part owing to its ability to mediate leukocyte recruitment.

α4β1- and α6β 1-integrins are functional receptors for midkine, a heparin-binding growth factor

Midkine is a heparin-binding growth factor that promotes the growth, survival, migration and differentiation of various target cells. So far, receptor-type protein tyrosine phosphatase ζ, low-density-lipoprotein-receptor-related protein and anaplastic lymphoma kinase have been identified as receptors for midkine. We found β1 integrin in midkine-binding proteins from 13-day-old mouse embryos. β1-Integrin bound to a midkine-agarose column and was eluted mostly with EDTA. Further study revealed that the α-subunits capable of binding to midkine were α4 and α6. Purified α4β1- and α6β1-integrins bound midkine. Anti-α4 antibody inhibited the midkine-dependent migration of osteoblastic cells, and anti-α6 antibody inhibited the midkine-dependent neurite outgrowth of embryonic neurons. After midkine treatment, tyrosine phosphorylation of paxillin, an integrin-associated molecule, was transiently increased in osteoblastic cells. Therefore, we concluded that α4β1- and α6β1-integrins are functional receptors for midkine. We observed that the low-density-lipoprotein-receptor-related-protein-6 ectodomain was immunoprecipitated with α6β1-integrin and α4β1-integrin. The low-density-lipoprotein-receptor-related-protein-6 ectodomain was also immunoprecipitated with receptor-type protein tyrosine phosphatase ζ. α4β1- and α6β1-Integrins are expected to co-operate with other midkine receptors, possibly in a multimolecular complex that contains other midkine receptors.

Preoperative serum midkine concentration is a prognostic marker for esophageal squamous cell carcinoma

High preoperative serum midkine concentration is associated with poor survival in patients with esophageal cancer, even after radical surgery, and thus may have prognostic value. Midkine (MK), a heparin‐binding growth factor, is expressed in numerous cancer tissues, and serum MK (S‐MK) concentrations are increased in patients with various neoplasms. The aim of this study is to evaluate the clinical significance of S‐MK in patients with esophageal squamous cell cancer (SCC). S‐MK was measured by enzyme‐linked immunosorbent assay in 135 healthy controls, 16 patients with benign esophageal disease, and 93 patients with primary esophageal SCC before surgery. The serum concentrations of carcinoembryonic antigen (CEA), SCC antigen (SCC‐Ag), and cytokeratin 19 fragment (CYFRA21–1) were also evaluated. All patients with esophageal SCC underwent radical esophagec‐tomy. Tumor MK expression was assessed by immunohistochemistry in 14 fresh tumor specimens. To determine whether S‐MK is of value as a prognostic factor, the authors conducted a survival analysis using Coxs proportional hazards model. S‐MK values in patients with esophageal SCC were significantly higher than those in healthy controls (417±342 pg/ml vs. 154±76 pg/ml, P<0.001). Using 300 pg/ml as the cut‐off value (representing the mean +2 standard deviations of the S‐MK of healthy controls), 61% of patients with esophageal SCC were classified as positive. MK expression by the tumor was significantly associated with high level of S‐MK. High S‐MK (≥300 pg/ml) was associated with tumor size, immunoreactivity and poor survival. Multivariate analysis indicated that S‐MK was an independent prognostic factor. S‐MK may be a useful tumor marker for esophageal SCC. Increased preoperative S‐MK in patients with esophageal SCC is associated with poor survival.

Midkine Plays a Protective Role Against Cardiac Ischemia/Reperfusion Injury Through a Reduction of Apoptotic Reaction

Background— Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, eg, neural survival, carcinogenesis, and tissue repair. MK could have a protective action against ischemia/reperfusion (I/R) injury in the heart, because MK was shown to have cytoprotective activity in cultured neurons and tumor cells. We investigated this hypothesis in mice with and without genetic MK deletion. Methods and Results— Myocardial injury after I/R was produced by transient occlusion of coronary arteries. In wild-type (Mdk+/+) mice, MK expression was increased after I/R in the periinfarct area. Infarct size/area at risk 24 hours after I/R in MK-deficient (Mdk−/−) mice was larger than in Mdk+/+ mice (55.4±9.1% versus 32.1±5.3%, P<0.05). Terminal dUTP nick end-labeling–positive myocyte population in the periinfarct area in Mdk−/− mice was higher than in Mdk+/+ mice (6.8±0.9% versus 3.2±0.6%, P<0.05). Left ventricular fractional shortening 24 hours after I/R in Mdk−/− mice was significantly less than that in Mdk+/+ mice (34.3±4.4% versus 50.8±2.1%, P<0.05). Supplemental application of MK protein to left ventricle of Mdk−/− mice at the time of I/R resulted in reduction of the infarct size. Application of exogenous MK to cultured cardiomyocytes resulted in increased Bcl-2 expression and decreased apoptosis after hypoxia/reoxygenation. Conclusions— These results suggest that MK plays a protective role against I/R injury, most likely through a prevention of apoptotic reaction. MK is a potentially important new molecular target for treatment of ischemic heart disease.

Protein tyrosine phosphatase receptor type Z is inactivated by ligand-induced oligomerization

Receptor‐type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPζ/RPTPβ), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G protein‐coupled receptor kinase‐interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand‐induced oligomerization.

Receptor-type protein tyrosine phosphatase ζ as a component of the signaling receptor complex for midkine-dependent survival of embryonic neurons

Midkine (MK), a heparin-binding growth factor, suppresses apoptosis of embryonic neurons in culture, induced by serum deprivation. Receptor-type protein tyrosine phosphatase zeta (PTP zeta) is a chondroitin sulfate proteoglycan with a transmembrane domain and intracellular tyrosine phosphatase domains. The activity of MK was abolished by digestion with chondroitinase ABC, or addition of the antibody to PTP zeta, while digestion with heparitinase showed no significant effect. These results suggested that the survival-promoting signal of MK was received by a receptor complex containing PTP zeta. Low density lipoprotein receptor-related protein (LRP) has been identified as another component of the signaling receptor. Ectodomains of two related proteins expressed on neurons, namely LRP6 and apoE receptor 2, were FLAG-tagged and examined for MK binding, using MK-agarose column. Both the ectodomains were found to exhibit calcium-dependent binding to MK. These proteins may participate in MK signaling in certain cases. The survival-promoting activity of MK was abolished by PP1, an inhibitor of src protein kinase, pertussis toxin, an inhibitor of G protein-linked signaling and sodium orthovanadate, an inhibitor of PTPs.

Correlation of elevated level of blood midkine with poor prognostic factors of human neuroblastomas.

The heparin-binding growth factor midkine (MK) is the product of a retinoic acid-responsive gene, and is implicated in neuronal survival and differentiation, and carcinogenesis. We previously reported that MK mRNA expression is elevated in neuroblastoma specimens at all stages, whereas pleiotrophin, the other member of the MK family, is expressed at high levels in favourable neuroblastomas. As MK is a secretory protein, it can be detected in the blood. Here, we show a significant correlation of the plasma MK level with prognostic factors of neuroblastomas. The plasma MK level was determined in 220 patients with neuroblastomas, and compared with that in children without malignant tumors (n=17, <500 pg ml−1). The plasma MK level became significantly elevated with advancing stages (stage 1: 445 pg ml−1 (median), n=73; stage 2: 589, n=39; stage 3: 864, n=40; stage 4: 1445, n=56; and stage 4S: 2439, n=12). More importantly, a higher MK level was strongly correlated with poor prognostic factors: over 1 year of age (P=0.0299), MYCN amplification (P<0.0001), low TrkA expression (P=0.0005), nonmass screening, sporadic neuroblastomas (P<0.0001), and diploidy/tetraploidy (P=0.0007). Thus, these results demonstrate that the plasma MK level is a good marker for evaluating the progression of neuroblastomas. Moreover, considering the ability of antisense MK oligodeoxyribonucleotide to suppress tumour growth of colorectal carcinoma cells in nude mice, as recently reported, the present study suggests that MK is a possible candidate molecular target for therapy for neuroblastomas.

Synthesis and characterization of oligomeric, anionic thiomalato-silver(I) complexes with biological activities

Abstract A water-soluble, thermally and light-stable, anionic AgI complex with a trianion of thiomalic acid, TMA3− (H3TMA = HOOCCH(SH)CH2COOH) as a ligand was prepared and isolated as a typically yellow-coloured powder of the sodium salt, which showed excellent antimicrobial activity against some bacteria, yeast and moulds. All attempts at crystallization were unsuccessful. This compound was characterized by complete elemental analyses, TG/DTA, FT-IR, various NMR (1H, 13C and 109Ag) spectroscopies, and molecular weight determination in aqueous solution by a combination of molmass measurement based on the cryoscopic method with degree of dissociation determination based on the [Na+] measurement in the equilibrium state using an Na+ ion-selective electrode. It was shown that this complex was not a monomeric species, but an oligomer with a formula of [NaH [Ag(TMA)]·0.5H2O]n(n = 15–19; MW = 4320–5470). Analogues of this anionic AgI complex were also obtained as potassium, barium, ammonium, lithium and caesium salts, as well as a free-acid form.

Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils

Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct. To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils. MK administration at the dose of 0.5-2 microg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult. The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult. MK administration at 2 h after occlusion did not ameliorate the neuronal death. These findings suggested that the therapeutic time window was narrow. The two to four times repeated administration of 2 microg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone. These findings suggested that the therapeutic time window was relatively narrow. The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.