Midkine Levels Research Articles

Expression of midkine in serum of patients with breast cancer and its clinical significance

Objective To investigate the serum midkine (MK) level in patients with breast cancer and its clinical significance.Methods The serum level of midkine was measured by using enzymelinked immunoadsobent assay in 100 healthy controls,50 cases of benign breast disease,and 120 cases of breast cancer before and after Surgery.Results The expression rate of midkine in serum of patients with breast cancer (65.52％) was significantly higher than in patients with benign breast disease (6.00％) and healthy controls (4.00％) (both P ＜0.01).The level of midkine in serum of patients with breast cancer before surgery [1 372 (896,324 6) ng/L] was significantly higher than that in patients with breast cancer after surgery [784 (563,890) ng/L] or breast benign disease [465 (420,574) ng/L] or healthy controls [450 (400,554) ng/L] (all P ＜0.01).The level of midkine in serum in patients with tumor recurrence [2 358 (1 123,4 787) ng/L] was obviously higher than that in patients with breast cancer before surgery (P ＜0.01).The preoperative level of midkine in serum of breast cancer patients was associated with clinical TNM stage,histological grade,axillary lymph nodes metastases and tumor size.Conclusion Surgery,clinical stages,histological grades,lymph nodes metastases and tumor recurrence were associated with the level of midkine in serum.Serum midkine level can be applied in the diagnosis and monitoring of breast cancer. Key words: Breast cancer; Midkine

Midkine expression is associated with clinicopathological features and BRAF mutation in papillary thyroid cancer

The objective of this study is to detect the expression of midkine (MK) in papillary thyroid cancer (PTC) and to evaluate whether MK expression is associated with clinicopathological features and BRAF mutation in PTC. The expression of MK in samples from 200 cases of PTC, 60 cases of adenomatoid nodule of thyroid, and 40 samples of tumor-adjacent normal thyroid tissue were assessed with immunohistochemistry. The BRAF mutation was detected by direct sequencing. The relationships between MK expression and the clinicopathological features of PTC and BRAF mutation were analyzed. The results demonstrated that MK was not expressed in tumor-adjacent normal tissue. The positive expression rates and MK scores were both higher in PTC than in adenomatoid nodule (positive expression rates: 88 vs. 8.3 %, P < 0.001; MK scores: 2.02 ± 0.93 vs. 0.08 ± 0.28, P < 0.001). The expression level of MK in PTC with extrathyroidal invasion, lymph node metastasis, or stage III/IV was significantly higher than that in PTC without such biological features (all P < 0.01). The overall prevalence of BRAF mutation was 66.5 % in PTC. The expression level of MK in PTC with BRAF mutation was significantly higher than that in PTC with wild-type BRAF (P < 0.001). We can conclude that MK is specifically expressed in PTC tissues and is associated with clinicopathological features and BRAF mutation. MK may be a helpful diagnostic and prognostic marker for PTC.

The Effects of Pulsed Electromagnetic Field (PEMF) on Osteoblast-Like Cells Cultured on Titanium and Titanium-Zirconium Surfaces

Commercially pure Ti, together with Ti Ni, Ti-6Al-4V, and Ti-6Al-7Nb alloys, are among the materials currently being used for this purpose. Titanium-zirconium (TiZr) has been developed that allows SLActive surface modification and that has comparable or better mechanical strength and improved biocompatibility compared with existing Ti alloys. Furthermore, approaches have targeted making the implant surface more hydrophilic, as with the Straumann SLActive surface, a modification of the SLA surface. The aim of this study is to evaluate the effects of pulsed electromagnetic field (PEMF) to the behavior of neonatal rat calvarial osteoblast-like cells cultured on commercially pure titanium (cpTi) and titanium-zirconium alloy (TiZr) discs with hydrophilic surface properties. Osteoblast cells were cultured on titanium and TiZr discs, and PEMF was applied. Cell proliferation rates, cell numbers, cell viability rates, alkaline phosphatase, and midkine (MK) levels were measured at 24 and 72 hours. At 24 hours, the number of cells was significantly higher in the TiZr group. At 72 hours, TiZr had a significantly higher number of cells when compared to SLActive, SLActive + PEMF, and machine surface + PEMF groups. At 24 hours, cell proliferation was significantly higher in the TiZr group than SLActive and TiZr + PEMF group. At 72 hours, TiZr group had higher proliferation rate than machine surface and TiZr + PEMF. Cell proliferation in the machine surface group was lower than both SLActive + PEMF and machine surface + PEMF. MK levels of PEMF-treated groups were lower than untreated groups for 72 hours. Our findings conclude that TiZr surfaces are similar to cpTi surfaces in terms of biocompatibility. However, PEMF application has a higher stimulative effect on cells cultured on cpTi surfaces when compared to TiZr.

The Signal Peptide of the Tumor-shared Antigen Midkine Hosts CD4+ T Cell Epitopes

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

Structure–function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis

Midkine is a heparin-binding di-domain growth factor, implicated in many biological processes as diverse as angiogenesis, neurogenesis and tumorigenesis. Elevated midkine levels reflect poor prognosis for many carcinomas, yet the molecular and cellular mechanisms orchestrating its activity remain unclear. At the present time, the individual structures of isolated half domains of human midkine are known and its functionally active C-terminal half domain remains a popular therapeutic target. In the present study, we determined the structure of full-length zebrafish midkine and show that it interacts with fondaparinux (a synthetic highly sulfated pentasaccharide) and natural heparin through a previously uncharacterized, but highly conserved, hinge region. Mutating six consecutive residues in the conserved hinge to glycine strongly abates heparin binding and midkine embryogenic activity. In contrast with previous invitro studies, we found that the isolated C-terminal half domain is not active invivo in embryos. Instead, we have demonstrated that the N-terminal half domain is needed to enhance heparin binding and mediate midkine embryogenic activity surprisingly in both heparin-dependent and -independent manners. Our findings provide new insights into the structural features of full-length midkine relevant for embryogenesis, and unravel additional therapeutic routes targeting the N-terminal half domain and conserved hinge.

Tumor location determines midkine level and its association with the disease progression in colorectal cancer patients: a pilot study

PurposeThe purpose of this study was to evaluate midkine, multipotential cytokine, and growth factor in colorectal cancer (CRC) stratified by tumor location.MethodsMidkine was assessed immunoenzymatically in paired cancerous and noncancerous tissues from 53 CRCs and referred to CRC stage, tumor location, and size, and circulating cytokine levels.ResultsMidkine was higher in cancerous versus noncancerous tissue in 98 % cases (424.2 vs. 31.1 pg/mg, p < 0.0001). Mean fold increase was 30.1; in 72.5 %, the relative increase was over fivefold. Midkine upregulation was more pronounced in colon than in rectum (fold increase: 36.6 vs. 12.7, p = 0.005) due to higher midkine level in noncancerous rectal than colonic tissue (45.5 vs. 26.2 pg/mg, p = 0.074). Tumor location affected midkine association with CRC stage. Midkine fold change was higher in advanced stages of rectal cancers (16.8 vs. 5.3, respectively in III/IV vs. I/II, p = 0.013), while it tended to be lower in colonic ones (25.3 vs. 47.8, p = 0.134). In addition, fold change in midkine level was higher in rectal N1 than N0 cancers (17.3 vs. 16.5, p = 0.032), while it tended to be lower in colonic cancers (23.6 vs. 50.1, p = 0.085). Midkine negatively correlated with tumor size (r = 0.40, p = 0.017), while it tended to positively correlate with its serum levels (r = 0.45, p = 0.081).ConclusionsMidkine is differently expressed in tumors arising from colonic and rectal mucosa, where it may play diverse roles in carcinogenesis. High midkine expression in noncancerous rectal mucosa might contribute to, a characteristic for rectal cancers, higher incidence of local recurrence. Divergent expression of midkine and its association pattern ought to be taken into account while designing midkine-directed therapies for CRC.

Midkine in the pathology of cancer, neural disease, and inflammation

Midkine (MK) is a heparin-binding growth factor involved in various cellular processes such as cellular proliferation, survival, and migration. In addition to these typical growth factor activities, MK exhibits several other activities related to fibrinolysis, blood pressure, host defense and other processes. Many cell-surface receptors have been identified to account for the multiple biological activities of MK. The expression of MK is frequently upregulated in many types of human carcinoma. Moreover, blood MK levels are closely correlated with patient outcome. Knockdown and blockade of MK suppress tumorigenesis and tumor development. Thus, MK serves as a tumor marker and a molecular target for cancer therapy. Furthermore, there is growing evidence that MK plays pivotal roles in neural and inflammatory diseases. Understanding of the mechanisms of action of MK is expected to create new therapeutic options for several human diseases.

The heparin binding growth factors midkine and pleiotrophin regulate the antinociceptive effects of morphine through α2-adrenergic independent mechanisms

Genetic deletion of pleiotrophin (PTN) impairs spinal nociceptive transmission suggesting that this heparin binding growth factor could play roles in acute pain processing. Despite the high functional redundancy between PTN and midkine (MK), the only other member of this family of growth factors, we now demonstrate that genetic inactivation of MK does not alter acute nociceptive transmission since pain responses of female MK genetically deficient (MK−/−) and wild type (WT+/+) mice were found to be similar in the hot-plate and tail-immersion tests. It has also been shown that morphine administration significantly regulates MK levels within the brain, suggesting that MK could play a role in morphine-induced antinociceptive effects. To test this hypothesis, we have now studied morphine-induced antinociceptive effects in female MK−/− and WT+/+ mice. We did not find differences among genotypes using different doses of morphine (2.5, 5 and 10mg/kg) in the hot-plate test. In contrast, we found that morphine significantly delayed pain responses in MK−/− mice compared to WT+/+ mice in the tail-immersion test. In confirmation of previous results from our group, we also found significantly enhanced morphine-induced antinociceptive effects in PTN−/− mice in the tail-immersion test. In addition, we now demonstrate that enhanced morphine analgesic effects in PTN−/− and MK−/− mice are not caused by a different contribution of descending noradrenergic inhibitory pathways since the α2-adrenergic antagonist yohimbine failed to alter morphine-induced analgesia in all genotypes. The data demonstrate that MK is an endogenous modulator of morphine antinociceptive effects, identify significant differences between PTN and MK in the control of pain processing at the spinal level, and support the hypothesis that inhibitors of the PTN/MK signaling pathway could potentiate opioid analgesia which may be relevant in opioid-refractory pain cases.

Imatinib mesylate decreases the cytotoxic effect of roscovitine on human glioblastoma cells in vitro and the role of midkine

The purpose of the present study was to overcome resistance to imatinib (IM) by combining it with roscovitine (ROSC) and to investigate whether or not midkine (MK) had an effect on this combination in the treatment of glioblastoma (GBL). Human T98 GBL cells were used to evaluate the effects of IM (10 μM), ROSC (200 μM) and their combination on the cell proliferation index, apoptotic index, the apoptotic protein and anti-apoptotic protein levels, and ultrastructure. All applications decreased the cell proliferation index and increased the apoptotic index, but ROSC was the most efficient drug and the second most efficient drug was IM. Notably, ROSC increased anti-apoptotic proteins levels (PDGFR-α, AQP-4, hTERT), COX-1 activity and ribosome numbers. The effects of ROSC on hTERT, MK, AQP-4 and MRP-1 levels and COX-1 activity were reported for the first time. ROSC induced the highest increase in caspase-3 levels. Autophagy was not involved in the activity of ROSC in GBL spheroids. The combination of IM with ROSC showed an antagonist effect in the treatment of human GBL cells. The combination group decreased certain anti-apoptotic protein levels (PDGFR-α, EGFR, p-gp, MRP-1 and MK), cAMP levels, COX-1 activity and apoptotic protein levels (caspase-3). However, it induced the highest increase in hTERT levels and COX-2 activity. Ribosome numbers were much lower than those in the ROSC group and no autophagic vacuole was observed. In conclusion, more investigations are required to identify the key regulatory components that are responsible for this antagonism; however, the determination of this combination therapy as a failure therapy may be precautionary for oncologists in the treatment of GBL patients and potentially may contribute to the efficacy of new therapeutic regimens.

Serum Midkine as a Prognostic Biomarker for Patients With Hepatocellular Carcinoma

Gene expression profiles of paired hepatocellular carcinoma (HCC) and adjacent noncancerous liver tissue samples revealed preferential expression of midkine in HCC. This finding suggested the clinical usefulness of midkine measurement in serum for monitoring HCC treatment response, recurrence, and progression. A prospective study in 285 patients, 144 in complete remission and 141 at risk for developing de novo HCC, was conducted. The changes in serum midkine level were in parallel with disease activity in about 81% of patients with HCC. The study also revealed that rapidly rising serum midkine levels occurred in patients in the terminal stage of HCC. The rising rate of serum midkine levels was inversely correlated with remaining survival days. However, serum midkine measurement did not detect emergence of new HCC in most patients in complete remission and in high-risk people without a history of HCC. Serum midkine levels can be useful to monitor HCC progression, and a sharp rise signals the approach of end of life in patients with HCC.

Midkine Exacerbates Pressure Overload-induced Cardiac Hypertrophy and Remodeling in Transgenic Mice

Background: Midkine (MK), a heparin-binding growth factor, has various functions such as migration of inflammatory cell and anti-apoptotic effect. We reported serum MK levels were increased in patients with heart failure and were independently associated with adverse cardiac events. The aim of this study was to examine the role of MK in cardiac hypertrophy and remodeling. Methods and Results: We generated transgenic mice overexpressing MK (MK-Tg) in a heart-specific manner. Transverse aortic constriction (TAC) or sham operation were performed in MK-Tg and wild-type (WT) mice.

English

Midkine (MK) is a heparin-binding growth factor and was found to be highly expressed in many types of human carcinomas. MK may become a novel tumor marker.&nbsp;In this study, we used&nbsp;the rabbit specific antibodies against human MK to establish a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) of MK system and applied it to detect serum MK levels in different types of cancer patients. The standard curve, precision and recovery rate were tested, respectively, and serum MK concentration of 102 cancers patients and 102 normal individuals were detected using this method. The detection range of this method was 0.2 to 10 ng/ml (R2&nbsp;= 0.97). The average intra and intro-assay&nbsp;coefficients of variation&nbsp;(CVs) were 3.6 and 7.9%, respectively. The average recovery rate was 89.9% when some standard antigens were added into the serum. The medians (25th and 75th percentiles) of serum MK levels were 1.35 ng/ml (0.96 and 1.64) in cancer patients and 0.30 ng/ml (0.23 and 0.38) in the controls; the MK levels of the patients were significantly higher than those of the controls (P&lt;0.05). Moreover, 87.2% of the patients showed more than 0.6 ng/ml levels of MK. Serum MK could serve as a general tumor marker with a good potential for clinical application. &nbsp; Key words:&nbsp;Midkine, rabbit monoclonal antibody, sandwich ELISA, tumor marker.

Midkine, A Multifunctional Cytokine, in Patients with Severe Sepsis and Septic Shock

The objective of the study was to evaluate whether severe sepsis and septic shock are related to alterations in midkine concentrations, to identify disease-related factors associated with these alterations, and to initially appraise whether midkine might serve as a biomarker in sepsis. Prospective observational cross-sectional study with 5-day follow-up. Circulating midkine was measured (enzyme-linked immunosorbent assay) in 38 septic (13 with severe sepsis, 25 with septic shock), 82 active inflammatory bowel disease (IBD) (26 with systemic inflammatory response syndrome [SIRS]) patients, and 87 healthy subjects. Midkine significantly increased along with a sequence: health-inflammation (IBD)-systemic inflammation (IBD-SIRS)-severe sepsis/septic shock. High midkine levels (>1,000 ng/L) were found in 63% of septic and in 19% of IBD-SIRS patients, whereas extremely high concentrations (>5,000 ng/L) were found in 16% vs. 4%. Although not different at admission, midkine gradually decreased in severe sepsis and remained high in shock. Similarly, persistently high midkine was observed in patients with cardiovascular insufficiency (CVI) and in mechanically ventilated as compared with normalizing levels in patients without CVI and not requiring ventilation. The differences in devised simple rates (Δ5th-1st) were significant in all these cases. Accordingly, admission midkine was higher in patients with metabolic acidosis. Concerning pathogen, gram-positive infections were associated with the highest midkine levels. In conclusion, sepsis and septic shock are associated with midkine elevation, substantially more pronounced than in inflammation, even systemic, revealing a new potential mediator of deregulation of neutrophil migration. Sepsis-related global hypoxia seems to contribute to midkine elevation. Our results substantiate further research on possible midkine application as a sepsis biomarker: in differentiating SIRS from sepsis and identifying gram-positive sepsis and septic patients at risk of CVI and shock.

Decreased therapeutic effects of noscapine combined with imatinib mesylate on human glioblastoma in vitro and the effect of midkine

BackgroundGlioblastoma (GBM) develops resistance to the advances in chemotherapy leading to poor prognosis and life quality. Consequently, new treatment modalities are needed. Our aims were to investigate the effects of combined noscapine (NOS) and imatinib mesylate (IM) on human GBM in vitro and the role of midkine (MK) in this new combination treatment.MethodsMonolayer and spheroid cultures of T98G human GBM cell line were used to evaluate the effects of IM (10 μM), Nos (10 μM) and their combination on cell proliferation and apoptotic indexes, cell cycle, the levels of antiapoptotic MK, MRP-1, p170, PFGFR-α, EGFR, bcl-2 proteins, apoptotic caspase-3 levels, morphology (SEM) and ultrastructure (TEM) for 72 hrs. Results were statistically analyzed using the Student's t-test.ResultsThe combination group induced highest decrease in cell proliferation and apoptotic indexes, caspase-3 levels, MRP-1 and PDGFR-α levels. The decrease in p170 levels were lower than IM but higher that NOS. The highest increases were in EGFR, MK, bcl-2 and cAMP levels in the combination group. The G0+G1 cell cycle arrest at the end of 72nd hr was the lowest in the combination group. Apoptotic appearence was observed rarely both in the morphologic and ultrastructural evaluation of the combination group. In addition, autophagic vacuoles which were frequently observed in the IM group were observed rarely.ConclusionsThe combination of Nos with IM showed antagonist effect in T98G human GBM cells in vitro. This antagonist effect was correlated highly with MK levels. The effects of NOS on MRP-1, MK and receptor tyrosine kinase levels were firstly demonstrated in our report. In addition, we proposed that MK is one of the modulator in the switch of autophagy to cell death or survival/resistance.

Combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinomain vitro

ObjectiveThe aim of the study was to investigate whether lithium chloride and medroxyprogesterone acetate can potentiate the cytotoxicity of imatinib mesylate in human endometrial cancer in vitro and the effect of midkine in these therapies.MethodsImatinib mesylate (50 µM), lithium chloride (100 µM), medroxyprogesterone acetate (200 µM) and their combination were applied to monolayer and three dimensional cultures of human Ishikawa endometrial cancer for 72 hours. The cell proliferation index, apoptotic index, caspase-3 and midkine levels, cell cycle distributions in monolayer cultures and cell ultrastructure in spheroid cultures were evaluated. Results were statistically analyzed using the Student's t-test.ResultsAll drug applications inhibited cell proliferation (p<0.05), however the combination were the effective groups for 72 hours (p<0.05). Interestingly, although the loss of efficiency was seen higly seen every 24 hours at single applications, the inhibition rates of the combination groups were almost same for 72 hours. In concordance with these results, the apoptotic index, caspase-3 levels (p<0.05), cell morphology and ultrastructure damages were much higher in the combination groups. Imatinib mesylate induced S-phase arrest, however other groups induced G0+G1-phase arrest at 24 hours and all groups induced G0+G1 arrest at 72 hours (p<0.05). Imatinib mesylate and imatinib mesylate with medroxyprogesterone acetate induced highest decrease in midkine levels, respectively (p<0.05).ConclusionThe present study showed that the combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinoma in vitro and the inhibition of midkine involved in their mechanism of action against endometrium defense.

Serum Midkine Is Related to NYHA Class and Cystatin C in Heart Transplant Recipients

Heart transplantation is now the established method of therapy for end-stage heart failure, with significantly improved outcomes over recent years. However, an increasingly prevalent complication in this population is that chronic kidney disease appears to be generally associated with subclinical inflammation. Midkine is a heparin-binding growth factor with various functions ranging from cell growth and survival to angiogenensis, repair, and inflammation. Recently, serum midkine has been reported to be a novel marker of cardiac events in heart failure patients. The aim of this study was to assess midkine concentration in 134 heart transplant recipients in relation to kidney function and New York Heart Association (NYHA) class. Heart transplant recipients had significantly higher serum creatinine, urea, cholesterol, triglycerides, fasting glucose, white blood cell count, and serum midkine, and lower estimated glomerular filtration rate than the control group. Serum midkine levels rose together with advancing NYHA class. Serum midkine was related to kidney function, NT-proBNP, transferrin, and prednisone dose. Cystatin C and NT-proBNP class turn out to be predictors of midkine in heart transplant recipients. Midkine levels are dependent on heart and kidney function, and might also represent a surrogate marker of subclinical inflammation.

Role of midkine in cadmium-induced liver, heart and kidney damage

Accumulation of the widespread environmental toxin cadmium (Cd) in tissues results in toxicity. Cd, which can induce a broad spectrum of biological effects, is a toxic substance and is associated with inflammation and apoptosis. Midkine (MK) has fibrinolytic, antiapoptotic, transforming, angiogenetic and chemotactic activities. After Cd toxicity, we found increased MK expression in liver cells in an in vitro cell culture model. The aim of this study was to determine the possibility of relationship between tissue MK expression levels, tumor necrosis factor α(TNF-α) levels and apoptosis in a chronic Cd toxicity model in rats. Male Wistar rats were exposed to Cd at the dose of 15 parts per million (ppm) for 8 weeks. MK levels were measured in kidney, heart and liver tissue by enzyme-linked-immunosorbent assay (ELISA). MK messenger RNA (mRNA) expression was evaluated by RT-PCR. Tissue apoptosis level was evaluated with tissue caspase-3 activity levels. Accumulation of Cd in liver is higher than the kidney and heart. Cd-treated rats had significantly higher tissue TNF-α and caspase-3 levels when compared with the control rats (p < 0.001). MK mRNA and protein levels were also significantly upregulated in the Cd-treated group (p < 0.05, p < 0.001, respectively). When compared with apoptosis in tissues, it was more prominent in the liver than kidney and heart. MK level is found increased 3, 1.7 and 1.3× folds in liver, kidney and heart, respectively. Our results showed that chronic Cd administration induces inflammation and apoptosis in rat liver, kidney and heart. MK involved in damage mechanisms of Cd-induced tissues. Further studies will show the underlying mechanism of increased MK expression in Cd toxicity.

Midkine in the plasma as a novel breast cancer marker.

1142 Background: Midkine, a heparin-binding growth factor, is up-regulated in many types of cancer. The aim of this study is to measure plasma midkine levels in patients with breast cancer and to assess its clinical significance in breast cancer. Methods: We examined plasma midkine levels in 95 healthy volunteers, 11 patients with ductal carcinoma in situ (DCIS), 111 patients with primary invasive breast cancer without distant metastasis (PIBC) and 25 patients with distant metastatic breast cancer (MBC), using an automatic immunoasssay analyzer (TOSOH AIA system). Results: In PIBC, we studied the correlation between plasma midkine levels and clinicopathological factors. Immunoreactive midkine was detectable in the plasma of healthy volunteers, and a cut-off level of 750 pg/ml was established. In patients with breast cancer, plasma midkine levels were increased above normal values. These elevated levels of midkine were seen in 1 (9.1%) of 11 patients with DCIS, 36 (32.4%) of 111 patients with PIBC, and 16 (64.0%) of 25 patients with MBC. Increased levels of midkine were correlated with menopausal status (p=0.0497) and nuclear grade (p=0.0343) in PIBC. Cancer detection rates based on midkine levels were higher than those based on three conventional markers including CA15-3 (p<0.0001), CEA (p=0.0077) and NCCST-439 (p<0.0001). Detection rates of breast cancer using combination of two conventional tumor markers (CA15-3/CEA, CA15-3/NCCST-439 or CEA/NCCST-439) with midkine is significantly higher than those using combination of three conventional tumor markers. Conclusions: Midkine may be a useful novel tumor marker for detection of breast cancer, superior to conventional tumor markers. No significant financial relationships to disclose.

Enhancement of vinorelbine-induced cytotoxicity and apoptosis by clomipramine and lithium chloride in human neuroblastoma cancer cell line SH-SY5Y

The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.

Serum Midkine as a Predictor of Cardiac Events in Patients With Chronic Heart Failure

Background Midkine, a heparin-binding growth factor, has various functions such as migration of inflammatory cell and anti-apoptotic effect. Invasion of inflammatory cell and cardiomyocyte apoptosis are involved in development and progression of heart failure (HF). However, the relationship between midkine and HF has not been previously examined. Therefore, we examined clinical significance of serum midkine levels to determine the prognosis of HF patients. Methods and Results Serum levels of midkine were measured at admission in 216 consecutive patients hospitalized for HF and 60 control subjects. Patients were prospectively followed during a mean follow-up period of 653 ± 375 days with the end points of cardiac death and progressive HF requiring rehospitalization. Serum concentrations of midkine were significantly higher in patients with HF than in controls. Patients with cardiac events had significantly higher concentrations of midkine than those without cardiac events. Kaplan-Meier analysis revealed that cardiac event rates increased markedly as midkine levels rose. Furthermore in the multivariate analysis, after adjustment for age, gender ,and complications, midkine was the independent predictor of cardiac events. Conclusion Serum midkine levels are increased in HF patients, and midkine is a novel marker for risk stratifying HF patients.