S-nitrosated Human Serum Albumin Research Articles

Drug Delivery System for Refractory Cancer Therapy via an Endogenous Albumin Transport System.

A unique phenomenon in solid tumors, the enhanced permeability and retention (EPR) effect is now well known in the development of macromolecular anticancer therapy. However, cancers with low vascular permeability have posed a challenge for these EPR based therapeutic systems. An intrinsic vascular modulator, such as nitric oxide (NO), could augment the endogenous EPR effect. However, the most important aim has been to construct an effective NO delivery system for cancer. Since it is well known that human serum albumin is one of the most important endogenous NO transport proteins in human circulation, for more than a decade we have demonstrated that S-nitrosated human serum albumin dimer (SNO-HSA-Dimer) becomes an enhancer of the EPR effect. Here, we summarize the enhanced effect of SNO-HSA-Dimer on the anticancer effect of macromolecular anticancer drugs such as PEGylated liposomal doxorubicin (Doxil®). In C26-bearing mice with highly permeable vasculature, SNO-HSA-Dimer is able to increase more 3-fold the tumor accumulation of these anticancer drugs, thereby tripling their anticancer effects. Interestingly, the tumor accumulation of Doxil® in B16-bearing mice, which are characterized by a low permeable vasculature, increased more than 6-fold in the presence of SNO-HSA-Dimer, and the improved accumulation of Doxil® led to both increased survival and decreased tumor volume. These results strongly suggest that the more cancer is refractory, the more the SNO-HSA-Dimer could enhance the EPR effect via an endogenous albumin transport (EAT) system. Accordingly, we conclude that the EAT system is promising as a drug delivery system (DDS) strategy for refractory cancer therapy.

Glutathione depletion and dual-model oxygen balance disruption for photodynamic therapy enhancement

Photodynamic therapy (PDT) is a prospective approach to cure tumor diseases. However, tumor micro-environment is notably characterized with severe hypoxia and high expression of glutathione (GSH), which seriously limit its clinical application. Here, based on the characteristics of perfluorocarbon (PFC) to dissolve substantial oxygen (O2) and the sensitivity of reductive GSH to S-NO group, we designed GSH depletion and dual-model O2 supply strategies to promote PDT enhancement. The PFC nanoliposomes (FI@Lip) and biocompatible NO donor S-nitrosated human serum albumin (HSA-SNO) were combined to synergistically combat the obstacle of tumor micro-environment, reducing GSH concentration and increasing singlet oxygen (1O2) generation. In vitro, after irradiation with NIR laser, the PFC in FI@Lip dissolved more O2 to increase 1O2 generation. In addition, with co-delivery of HSA-SNO, it can effectively promote GSH depletion to recover 1O2 level and release NO concurrently to inhibit mitochondrial respiration. This combination strategy of FI@Lip and HSA-SNO obviously relieved intracellular hypoxia and decreased GSH to increase more toxic 1O2 generation for PDT enhancement. The present work will play as an enlightening role in PDT design and clinical application in the near future.

Dual Therapeutic Effects of an Albumin-Based Nitric Oxide Donor on 2 Experimental Models of Chronic Kidney Disease

Chronic kidney disease (CKD) is accompanied by a variety of complications, typically renal anemia and kidney fibrosis. Accordingly, it is desirable to develop the novel therapeutics that can treat these CKD conditions. Since nitric oxide (NO) has multiple functions including hypoxia inducible factor stabilizing, anti-inflammatory, anti-oxidative, and anti-apoptoic activities, the use of NO for the CKD therapy has attracted considerable interest. Here, we evaluate the therapeutic impacts of S-nitrosated human serum albumin (SNO-HSA), a long-lasting NO donor, on 2 animal models of CKD. SNO-HSA increased the expression of erythropoietin (EPO), VEGF, and eNOS by stabilizing hypoxia inducible factor–1α in HepG2 and HK-2 cells. SNO-HSA increased hematopoiesis in both healthy and renal anemia rats, suggesting the promotion of EPO production. In unilateral ureteral obstruction–treated mice, SNO-HSA ameliorated kidney fibrosis by suppressing the accumulation of renal extracellular matrix. SNO-HSA also inhibited unilateral ureteral obstruction–induced α–smooth muscle actin increase and E-cadherin decrease, suggesting that SNO-HSA might suppress the accumulation of myofibroblasts, an important factor of fibrosis. SNO-HSA also inhibited the elevations of fibrosis factors, such as transforming growth factor–β, interleukin-6, and oxidative stress, while it increased EPO production, an anti-fibrosis factor. In conclusion, SNO-HSA has the potential to function as a dual therapeutics for renal anemia and kidney fibrosis.

Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albumin-protein interactions using S-nitrosated human serum albumin dimer.

In the latest trend of anticancer chemotherapy research, there were many macromolecular anticancer drugs developed based on enhanced permeability and retention (EPR) effect, such as albumin bound paclitaxel nanoparticle (nab- PTX, also called Abraxane®). However, cancers with low vascular permeability posed a challenge for these EPR based therapeutic systems. Augmenting the intrinsic EPR effect with an intrinsic vascular modulator such as nitric oxide (NO) could be a promising strategy. S-nitrosated human serum albumin dimer (SNO-HSA Dimer) shown promising activity previously was evaluated for the synergistic effect when used as a pretreatment agent in nab-PTX therapy against various tumor models. In the high vascular permeability C26 murine colon cancer subcutaneous inoculation model, SNO-HSA Dimer enhanced tumor selectivity of nab-PTX, and attenuated myelosuppression. SNO-HSA Dimer also augmented the tumor growth inhibition of nab-PTX in low vascular permeability B16 murine melanoma subcutaneous inoculation model. Furthermore, nab-PTX therapy combined with SNO-HSA Dimer showed higher antitumor activity and improved survival rate of SUIT2 human pancreatic cancer orthotopic model. In conclusion, SNO-HSA Dimer could enhance the therapeutic effect of nab-PTX even in low vascular permeability or intractable pancreatic cancers. The possible underlying mechanisms of action of SNO-HSA Dimer were discussed.

Albumin-Based Nitric Oxide Traffic System for the Treatment of Intractable Cancers

Biomacromolecules (>40 kDa) have been developed as drug delivery system (DDS) carriers of low-molecular weight drugs to promote these drugs' uptake by cancer tissues via enhanced permeability and retention (EPR) effects. Human serum albumin (HSA) has been found to accumulate in cancer tissues via this EPR effect. HSA is the most abundant protein in serum, which performs essential physiological functions such as the transportation of many endogenous and exogenous ligands. Nitric oxide (NO) is a very small ligand of HSA; it is a unique and diffusible molecular messenger that plays a central role in mammalian physiology. Although the in vivo half-life of NO is extremely short, HSA could prolong the half-life of NO via S-nitrosation at the position of Cys-34. S-Nitrosated HSA (mono-SNO-HSA) is called an 'Endogenous NO traffic protein,' due to the highly stable S-nitroso form in circulating blood, and to the efficiency of S-transnitrosation in cells that require NO. Mono-SNO-HSA possesses a very strong cytoprotective action via the induction of heme oxygenase-1. On the other hand, HSA reinforced with approximately seven NO molecules (poly-SNO-HSA), which we developed by means of chemical modification, possesses multiple anticancer activities. Our previous data clarified that the high expression of protein disulfide isomerase on the surface of cancer cells plays a very important role in the anticancer action of poly-SNO-HSA. In this review, we focus on the advantage of poly-SNO-HSA in treating intractable cancers from the viewpoint of drug delivery systems and drug resistance.

Human Serum Albumin as Carrier in Drug Delivery Systems

Recently, human serum albumin (HSA) has emerged as a versatile carrier for therapeutic agents against diabetes, cancer, and infectious diseases. Market-approved products include fatty acid derivatives of human insulin for diabetes and the paclitaxel-HSA nanoparticle for various cancers such as metastatic breast cancer and advanced pancreatic cancer. In this review, we focus on the next-generation approach including HSA-binding bioactive gas such as nitric oxide (NO) for treating ischemic/reperfusion injury, cancer, and bacterial infection. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated HSA (SNO-HSA), which is why we are investigating whether this albumin form can be therapeutically useful. Recently, we have developed SNO-HSA analogues such as SNO-HSA with many conjugated SNO groups (poly-SNO-HSA) prepared using chemical modification. Unexpectedly, we found striking inverse effects between poly-SNO-HSA and SNO-HSA. Despite the fact that SNO-HSA inhibits apoptosis, poly-SNO-HSA possesses very strong pro-apoptotic effects against tumor cells. Furthermore, poly-SNO-HSA can reduce or even completely eliminate the multidrug resistance often developed by cancer cells. In this review, we put forward the possibility that poly-SNO-HSA can be used as a safe, effective multifunctional antitumor agent.

S-nitrosated human serum albumin dimer as novel nano EPR enhancer applied to nano-anticancer drugs

S-nitrosated human serum albumin dimer as novel nano EPR enhancer applied to nano-anticancer drugs

S-Nitrosated human serum albumin dimer as novel nano-EPR enhancer applied to macromolecular anti-tumor drugs such as micelles and liposomes

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3–4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.

Poly-S-nitrosated human albumin enhances the antitumor and antimetastasis effect of bevacizumab, partly by inhibiting autophagy through the generation of nitric oxide.

Autophagy is one of the major causes of drug resistance. For example, the angiogenesis inhibitor bevacizumab shows only transient and short-term therapeutic effects, whereas long-term therapeutic benefits are rarely observed, probably due to hypoxia-induced autophagy. Nitric oxide (NO) is an important molecule with multiple functions, and it has recently been reported to function as a regulator of autophagy. Therefore, a reasonable therapeutic strategy for overcoming drug resistance by NO would involve it being directly delivered to the tumor. Here, we investigated the inhibitory effect of NO on autophagy by using a macromolecular NO donor S-nitrosated human serum albumin (SNO-HSA) with a high degree of NO loading and tumor targeting potential. In colon 26 (C26) cells, SNO-HSA significantly suppressed hypoxia-induced autophagy by inhibiting the phosphorylation of JNK1 and the expression of its downstream molecule Beclin1. The effect of SNO-HSA was also confirmed in vivo by combining it with Bev. In C26-bearing mice, significant suppression of tumor growth as well as lung metastasis was achieved in the combination group compared to the SNO-HSA or bevacizumab alone group. Similar to the in vitro experiments, the immunostaining of tumor tissues clearly showed that SNO-HSA inhibited the autophagy of tumor cells induced by bevacizumab treatment. In addition to other known antitumor effects of SNO-HSA, that is, the induction of apoptosis and the inhibition of multidrug efflux pumps, these data may open alternate strategies for cancer chemotherapy by taking advantage of the ability of SNO-HSA to suppress autophagy-mediated drug resistance and enhance the efficacy of chemotherapy.

UW solution improved with high anti-apoptotic activity by S-nitrosated human serum albumin

S-Nitrosated human serum albumin (SNO-HSA) is useful in preventing liver ischemia/reperfusion injury, and SNO-HSA should thus be able to prevent cell injury during liver transplantation. However, the potential protective effect of SNO-HSA on a combination of cold and warm ischemia, which is obligatory when performing liver transplantation, has not been examined. Therefore, we evaluated the protective effect of SNO-HSA added to University of Wisconsin (UW) solution during cold or/and warm ischemia in situ and in vitro. First, we observed that apoptotic and necrotic cell death were increased during cold and warm ischemia, respectively. SNO-HSA, which possesses anti-apoptosis activity at low NO concentrations, can inhibit cold ischemia injury both in situ and in vitro. In contrast, SNO-HSA had no significant effect on warm liver ischemia injury which, however, can be reduced by UW solution. We also demonstrated that the cellular uptake of NO from SNO-HSA can occur during cold ischemia resulting in induction of heme oxygenase-1 within 3h of cold ischemia. Our results indicate that treatment with SNO-HSA or UW solution alone is not sufficient to inhibit liver injury during a period of both cold and warm ischemia. However, a combination of SNO-HSA and UW solution can be used to prevent the two types of ischemia. SNO-HSA-added UW solution could be very useful in transplantation, because the previously imposed constraints on preservation time can be removed. This is a great advantage in a situation as the present one with increased utilization of scarce donor organs for more recipients.

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

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.

One-step preparation of S-nitrosated human serum albumin with high biological activities

S-Nitrosated human serum albumin (SNO-HSA) is a large molecular weight nitric oxide carrier in human plasma, and because of its many beneficial effects in different tests, it is currently under investigation as a cytoprotective agent. However, making SNO-HSA preparations is a complicated and time-consuming process. We found that binding of caprylic acid (CA) and N-acetyl- l-tryptophan (N-AcTrp) to defatted mercaptalbumin increased S-nitrosation by S-nitrosoglutathione (GS-NO) by making Cys-34 of HSA more accessible and by protecting it against oxidation, respectively. Fortunately, HSA solutions for clinical use contain high concentrations of CA and N-AcTrp as stabilizers. By making use of that fact it was possible to work-out a fast and simple procedure for producing SNO-HSA: incubation of a commercial HSA formulation with GS-NO for only 1 min results in S-nitrosation of HSA. The biological usefulness of such a preparation was tested in a rat ischemia–reperfusion liver injury model. Although our procedure for making SNO-HSA is fast and straightforward, the cytoprotective effect of the preparation was similar to, or better than, that of a preparation made in a more traditional way. The clinical development of SNO-HSA as a strong cytoprotective agent is under way using this method in collaboration with clinicians and industrial developers.

S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme.

An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine.