The Therapeutic Effect of Human Serum Albumin Dimer-Doxorubicin Complex against Human Pancreatic Tumors.

Human serum albumin (HSA) is a versatile drug carrier with active tumor targeting capacity for an antitumor drug delivery system. Nanoparticle albumin-bound (nab)-technology, such as nab-paclitaxel (Abraxane®), has attracted significant interest in drug delivery research. Recently, we demonstrated that HSA dimer (HSA-d) possesses a higher tumor distribution than HSA monomer (HSA-m). Therefore, HSA-d is more suitable as a drug carrier for antitumor therapy and can improve nab technology. This study investigated the efficacy of HSA-d-doxorubicin (HSA-d-DOX) as next-generation nab technology for tumor treatment. DOX conjugated to HSA-d via a tunable pH-sensitive linker for the controlled release of DOX. Lyophilization did not affect the particle size of HSA-d-DOX or the release of DOX. HSA-d-DOX showed significantly higher cytotoxicity than HSA-m-DOX in vitro. In the SUIzo Tumor-2 (SUIT2) human pancreatic tumor subcutaneous inoculation model, HSA-d-DOX could significantly inhibit tumor growth without causing serious side effects, as compared to the HSA binding DOX prodrug, which utilized endogenous HSA as a nano-drug delivery system (DDS) carrier. These results indicate that HSA-d could function as a natural solubilizer of insoluble drugs and an active targeting carrier in intractable tumors with low vascular permeability, such as pancreatic tumors. In conclusion, HSA-d can be an effective drug carrier for the antitumor drug delivery system against human pancreatic tumors.

S-Nitrosated alpha-1-acid glycoprotein exhibits antibacterial activity against multidrug-resistant bacteria strains and synergistically enhances the effect of antibiotics.

Alpha-1-acid glycoprotein (AGP) is a major acute-phase protein. Biosynthesis of AGP increases markedly during inflammation and infection, similar to nitric oxide (NO) biosynthesis. AGP variant A (AGP) contains a reduced cysteine (Cys149). Previously, we reported that S-nitrosated AGP (SNO-AGP) synthesized by reaction with a NO donor, possessed very strong broad-spectrum antimicrobial activity (IC50 = 10-9-10-6 M). In this study, using a cecal ligation and puncture animal model, we confirmed that AGP can be endogenously S-nitrosated during infection. Furthermore, we examined the antibacterial property of SNO-AGP against multidrug-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa to investigate the involvement of SNO-AGP in the host defense system. Our results showed that SNO-AGP could inhibit multidrug efflux pump, AcrAB-TolC, a major contributor to bacterial multidrug resistance. In addition, SNO-AGP decreased biofilm formation and ATP level in bacteria, indicating that SNO-AGP can revert drug resistance. It was also noteworthy that SNO-AGP showed synergistic effects with the existing antibiotics (oxacillin, imipenem, norfloxacin, erythromycin, and tetracycline). In conclusion, SNO-AGP participated in the host defense system and has potential as a novel agent for single or combination antimicrobial therapy.

A novel S-sulfhydrated human serum albumin preparation suppresses melanin synthesis.

Products of ultraviolet (UV) irradiation such as reactive oxygen species (ROS) and nitric oxide (NO) stimulate melanin synthesis. Reactive sulfur species (RSS) have been shown to have strong ROS and NO scavenging effects. However, the instability and low retention of RSS limit their use as inhibitors of melanin synthesis. The free thiol at Cys34 on human serum albumin (HSA) is highly stable, has a long retention and possess a high reactivity for RSS. We report herein on the development of an HSA based RSS delivery system. Sulfane sulfur derivatives released from sodium polysulfides (Na2Sn) react readily with HSA. An assay for estimating the elimination of sulfide from polysulfide showed that almost all of the sulfur released from Na2Sn bound to HSA. The Na2Sn-treated HSA was found to efficiently scavenge ROS and NO produced from chemical reagents. The Na2Sn-treated HSA was also found to inhibit melanin synthesis in B16 melanoma cells and this inhibition was independent of the number of added sulfur atoms. In B16 melanoma cells, the Na2Sn-treated HSA also inhibited the levels of ROS and NO induced by UV radiation. Finally, the Na2Sn-treated HSA inhibited melanin synthesis from L-DOPA and mushroom tyrosinase and suppressed the extent of aggregation of melanin pigments. These data suggest that Na2Sn-treated HSA inhibits tyrosinase activity for melanin synthesis via two pathways; by directly inhibiting ROS signaling and by scavenging NO. These findings indicate that Na2Sn-treated HSA has potential to be an attractive and effective candidate for use as a skin whitening agent.

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.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 µM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

Apoptosis induction of poly-S-nitrosated human serum albumin in resistant solid tumor under hypoxia can be restored by phosphodiesterase 5 inhibition.

Poly-S-nitrosated human serum albumin (Poly-SNO-HSA) delivered and accumulated nitric oxide (NO) in tumors and induces apoptosis. Tumor hypoxia is strongly associated with malignant progression and tumor resistance to therapy. In this study, we examined the cytotoxic effect of Poly-SNO-HSA under hypoxia on the murine colon 26 adenocarcinoma (C26) cells in vitro and in vivo. Under hypoxia, at about 4 times LD50 dose of Poly-SNO-HSA in vitro, the reactive oxygen species production was hindered but apoptotic cells were induced via cGMP pathway as the effect was suppressed by a soluble guanylate cyclase inhibitor, NS2028. The apoptosis induction effect of low dose Poly-SNO-HSA on C26 cells in vitro under hypoxia can be restored by a phosphodiesterase 5 (PDE5) inhibitor, vardenafil. In C26-bearing mice, Poly-SNO-HSA/vardenafil combination treatment significantly suppressed the tumor volume compared with Poly-SNO-HSA or vardenafil treatment alone. Furthermore, the core tumor tissues showed increased expression of caspase-3 than the non-core tissue. The expression of caspase-3 appeared to overlap with the hypoxic zone of tumor tissues. Similar results were also obtained when the experiments were repeated using Epimedium extract, a natural herbal supplement with PDE5 inhibition activity. In conclusion, Poly-SNO-HSA/PDE5 inhibitors combination therapy is a promising approach for enhancing the anticancer therapeutic effects of Poly-SNO-HSA against not only anti-cancer drug resistance but also hypoxic stress related solid tumor resistance.

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease.

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.

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.

A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy.

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin-Trx fusion protein (HSA-Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA-Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N-acetyl-ß-D-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA-Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA-Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA-Trx has the potential to effectively prevent CIN.