Toshiya Kai

S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding

Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation.

Design and Evaluation of S-Nitrosylated Human Serum Albumin as a Novel Anticancer Drug

In recent studies, the cytotoxic activity of NO has been investigated for its potential use in anticancer therapies. Nitrosated human serum albumin (NO-HSA) may act as a reservoir of NO in vivo. However, there are no published reports regarding the effects of NO-HSA on cancer. Therefore, the present study investigated the antitumor activity of NO-HSA. NO-HSA was prepared by incubating HSA, which had been sulfhydrylated using iminothiolane, with isopentyl nitrite (6.64 mol NO/mol HSA). Antitumor activity was examined in vitro using murine colon 26 carcinoma (C26) cells and in vivo using C26 tumor-bearing mice. Exposure to NO-HSA increased the production of reactive oxygen species in C26 cells. Flow cytometric analysis using rhodamine 123 showed that NO-HSA caused mitochondrial depolarization. Activation of caspase-3 and DNA fragmentation were observed in C26 cells after incubation with 100 μM NO-HSA for 24 h, and NO-HSA inhibited the growth of C26 cells in a concentration-dependent manner. The growth of C26 tumors in mice was significantly inhibited by administration of NO-HSA compared with saline and HSA treatment. Immunohistochemical analysis of tumor tissues demonstrated an increase in terminal deoxynucleotidyl transferase dUTP nickend labeling-positive cells in NO-HSA-treated mice, suggesting that inhibition of tumor growth by NO-HSA was mediated through induction of apoptosis. Biochemical parameters (such as serum creatinine, blood urea nitrogen, aspartate aminotransferase, and alanine aminotransferase) showed no significant differences among the three treatment groups, indicating that NO-HSA did not cause hepatic or renal damage. These results suggest that NO-HSA has the potential for chemopreventive and/or chemotherapeutic activity with few side effects.

Pharmacokinetic study of enclosed hemoglobin and outer lipid component after the administration of hemoglobin vesicles as an artificial oxygen carrier.

The hemoglobin vesicle (HbV) is an artificial oxygen carrier that encapsulates a concentrated Hb solution in lipid vesicles (liposomes). The pharmacokinetic properties of HbV were investigated in mice and rats. With use of HbV in which the internal Hb was labeled with 125I (125I-HbV) and cell-free 125I-Hb, it was found that encapsulation of Hb increased the half-life by 30 times, accompanied by decreased distribution in both the liver and kidney. The half-life of HbV was increased, and the uptake clearance for the liver and spleen were decreased with increasing doses of HbV. In an in vitro study, the specific uptake and degradation of HbV in RAW 264.7 cells were found, but this was not the case for parenchymal and endothelial cells. The pharmacokinetics of HbV components (internal Hb and liposomal lipid) were also investigated using 125I-HbV and 3H-HbV (liposomal cholesterol was radiolabeled with tritium-3). The time courses for the plasma concentration curves of 125I-HbV, 3H-HbV, and iron derived from HbV suggest that HbV maintain an intact structure in the blood circulation up to 24 h after injection. 125I-HbV and 3H-HbV were distributed mainly to the liver and spleen. Internal Hb disappeared from both the liver and spleen 5 days after injection, and the liposomal cholesterol disappeared at approximately 14 days. Internal Hb was excreted into the urine and cholesterol into feces via biliary excretion. These results suggest that the HbV has a reasonable blood retention and metabolic and excretion performance and could be used as an oxygen carrier.

Pharmacokinetics of single and repeated injection of hemoglobin-vesicles in hemorrhagic shock rat model

Hemoglobin-vesicles (HbV) are liposomal artificial oxygen carriers that may be useful as a resuscitation fluid during hemorrhagic shock (HS). It is well-known that the pharmacokinetic properties of liposome change in response to both pathological conditions and repeated administration. Therefore, we compared the pharmacokinetics of single versus repeated administration of HbV during HS. HS was induced by withdrawal of 40% of total blood volume. The normal (non-HS) and HS1 group was received an injection of 125I-labeled HbV (125I-HbV). The HS2 group was resuscitated with non-labeled HbV, and 1 h later, it received an injection of 125I-HbV. The half-life was shorter in HS1 rats, but it returned to non-HS levels after the second HbV injection. During 12 h after administration of HbV, tissue distribution of HbV was greatest in the HS1 group; however, the HS2 group had the greatest tissue distribution at subsequent time points. Excretion into urine, major elimination pathway, did not differ between non-HS and HS1 rats, but was significantly reduced in the HS2 group. Furthermore, the half-life of HbV in humans was estimated to be approximately 3-4 days using an allometric equation. This suggests that HbV may be a useful artificial oxygen carrier in HS based on HbV pharmacokinetics.

S -nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.

Nitrosylated human serum albumin (SNO-HSA) induces apoptosis in tumor cells.

Recently, nitric oxide has been investigated as a potential anti-cancer therapy because of its cytotoxic activity. Previously, we found that S-nitrosylated human serum albumin (SNO-HSA) induced apoptosis in C26 cells, demonstrating for the first time that SNO-HSA has potential as an anti-cancer drug. In the present study, the anti-tumor activity of SNO-HSA in another tumor type of cancer cell was investigated using murine tumor LY-80 cells. Mitochondrial depolarization, activation of caspase-3 and DNA fragmentation were induced in LY-80 cells by SNO-HSA treatment in a dose-dependent manner. Inhibition of caspase activity resulted in complete inhibition of DNA fragmentation induced by SNO-HSA. The cytotoxic effects of SNO-HSA on LY-80 were concentration-dependent. Tumor growth in LY-80-tumor-bearing rats was significantly inhibited by administration of SNO-HSA compared with saline- and HSA-treatment. These results suggest that SNO-HSA has potential as a chemopreventive and/or chemotherapeutic agent because it induces apoptosis in tumor cells.

Effects of endogenous ligands on the biological role of human serum albumin in S-nitrosylation

Many proteins have been identified as targets for S-nitrosylation, including structural and signaling proteins, a nd ion channels. S-nitrosylation plays an important role in regulating their activity and function. We used human serum albumin (HS A), a major endogenous NO traffic protein, and studied the effect of mediators on S-nitrosylation processes which control NO bioactivity. By using NOC-7, S-nitrosoglutathione, and activated RAW264.7 cells as NO-donors we found that high-affinity binding of endogenous ligands (Cu(2+), bilirubin and fatty acid) can affect these processes. It is likely that the same effects take place in many clinical situations characterized by increased fatty acid concentrations in plasma such as type II diabetes and the metabolic syndrome. Thus, endogenous ligands, changing their plasma concentrations, could be a novel type of mediator of S-nitrosylation not only in the case of HSA but also for other target proteins.

Chromium(III) ion and thyroxine cooperate to stabilize the transthyretin tetramer and suppress in vitro amyloid fibril formation

Transthyretin (TTR) amyloid fibril formation, which is triggered by the dissociation of tetrameric TTR, appears to be the causative factor in familial amyloidotic polyneuropathy and senile systemic amyloidosis. Binding of thyroxine (T4), a native ligand of TTR, stabilizes the tetramer, but the bioavailability of T4 for TTR binding is limited due to the preferential binding of T4 to globulin, the major T4 carrier in plasma. Here, we show that Cr3+ increased the T4‐binding capacity of wild‐type (WT) and amyloidogenic V30M‐TTR. Moreover, we demonstrate that Cr3+ and T4 cooperatively suppressed in vitro fibril formation due to the stabilization of WT‐TTR and V30M‐TTR.

Cellular uptake mechanisms and responses to NO transferred from mono- and poly-S-nitrosated human serum albumin

Abstract Endogenous S-nitrosated human serum albumin (E-Mono-SNO-HSA) is a large molecular weight nitric oxide (NO) carrier in human plasma, which has shown many beneficial effects in different animal models. To construct more efficient SNO-HSA preparations, SNO-HSA with many conjugated SNO groups has been prepared using chemical modification (CM-Poly-SNO-HSA). We have compared the properties of such a preparation to those of E-Mono-SNO-HSA. Cellular uptake of NO from E-Mono-SNO-HSA partly takes place via low molecular weight thiol, and it results in cytoprotective effects by induction of heme oxygenase-1. By contrast, transfer of NO from CM-Poly-SNO-HSA into the cells is faster and more pronounced. The influx mainly takes place by cell-surface protein disulfide isomerase. The considerable NO inflow results in apoptotic cell death by ROS induction and caspase-3 activation. Thus, increasing the number of SNO groups on HSA does not simply intensify the cellular responses to the product but can also result in very different effects.

Elucidation of the therapeutic enhancer mechanism of poly-S-nitrosated human serum albumin against multidrug-resistant tumor in animal models.

Human serum albumin (HSA) is the most abundant circulating protein and its S-nitrosated form serves as a reservoir of nitric oxide (NO). Previously, we prepared poly-S-nitrosated HSA (Poly-SNO-HSA) by incubation with Trauts Reagent and isopentyl nitrite and evaluated its potential as a novel anticancer agent through apoptosis involving the caspase-3 pathway. Recently, NO donors such as nitroglycerin were reported to revert the resistance to anticancer agents. Therefore, now we have evaluated the effect of the above type of Poly-SNO-HSA on the resistance to doxorubicin (dx) in human myelogenous leukemic cells (K562 cells). P-gp expression and dx accumulation in K562 and dx-resistant K562 cells (K562/dx cells) were quantified using Western blot and FACS analysis, respectively. Compared with parent K562 cells, higher expression of P-gp and lower accumulation of dx were shown in K562/dx cells. Poly-SNO-HSA caused increased dx accumulation in K562/dx cells by decreasing the expressions of P-gp and HIF-1α. Other experiments with the guanylate cyclase inhibitor ODQ and 8-Br-cGMP revealed that also a cGMP signaling pathway is involved in the Poly-SNO-HSA induced increase in dx accumulation. Furthermore, in vivo studies showed that co-treatment with Poly-SNO-HSA enhanced the anticancer effect of dx in K562/dx cells-bearing mice. Thus, in addition to its proapoptotic effect Poly-SNO-HSA can in an efficient manner revert drug resistance both in vitro and in vivo, and two pathways for this effect have been identified.