Use of pH-Active Catechol-Bearing Polymeric Nanogels with Glutathione-Responsive Dissociation to Codeliver Bortezomib and Doxorubicin for the Synergistic Therapy of Cancer.

Synergistic therapy holds promising potential in cancer treatment. Here, the inclusion of catechol moieties, a disulfide cross-linked structure, and pendent carboxyl into the network of polymeric nanogels with glutathione (GSH)-responsive dissociation and pH-sensitive release is first disclosed for the codelivery of doxorubicin (DOX) and bortezomib (BTZ) in synergistic cancer therapy. The pendent carboxyl groups and catechol moieties are exploited to absorb DOX through electrostatic interaction and conjugate BTZ through boronate ester, respectively. Both electrostatic interactions and boronate ester are stable at neutral or alkaline pH, while they are instable in an acidic environment to further recover the activities of BTZ and DOX. The polymeric nanogels possess a superior stability to prevent the premature leakage of drugs in a physiological environment, while their structure is destroyed in response to a typical endogenous stimulus (GSH) to unload drugs. The dissociation of the drug-loaded nanogels accelerates the intracellular release of DOX and BTZ and further enhances the therapeutic efficacy. In vitro and in vivo investigations revealed that the dual-drug loaded polymeric nanogels exhibited a strong ability to suppress tumor growth. This study thus proposes a new perspective on the production of multifunctional polymeric nanogels through the introduction of different functional monomers.

Benextramine and derivatives as novel human monoamine oxidases inhibitors: an integrated approach.

The two human monoamine oxidase isoforms (namely MAO A and MAO B) are enzymes involved in the catabolism of monoamines, including neurotransmitters, and for this reason are well-known and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases, for which novel pharmacological approaches are necessary. Benextramine is a tetraamine disulfide mainly known as irreversible α-adrenergic antagonist, but able to hit additional targets involved in neurodegeneration. As the molecular structures of monoamine oxidases contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds were found to induce irreversible inactivation of MAOs with inactivation potency depending on both the polyamine structure and the enzyme isoform. The more effective compounds generally showed preference for MAO B. Structure-activity relationships studies revealed the key role played by the disulfide core of these molecules in the inactivation mechanism. Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of this type of inhibitors thus suggesting that their covalent binding inside the MAO active site sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line. These results demonstrated for the first time that benextramine and its derivatives can inactivate human MAOs exploiting a mechanism different from that of the classical MAO inhibitors and could be a starting point for the development of pharmacological tools in neurodegenerative diseases.

Biomimetic engineering endothelium-like coating on cardiovascular stent through heparin and nitric oxide-generating compound synergistic modification strategy.

Co-immobilization of two or more molecules with different and complementary functions to prevent thrombosis, suppress smooth muscle cell (SMC) proliferation, and support endothelial cell (EC) growth is generally considered to be promising for the re-endothelialization on cardiovascular stents. However, integration of molecules with distinct therapeutic effects does not necessarily result in synergistic physiological functions due to the lack of interactions among them, limiting their practical efficacy. Herein, we apply heparin and nitric oxide (NO), two key molecules of the physiological functions of endothelium, to develop an endothelium-mimetic coating. Such coating is achieved by sequential conjugation of heparin and the NO-generating compound selenocystamine (SeCA) on an amine-bearing film of plasma polymerized allylamine. The resulting surface combines the anti-coagulant (anti-FXa) function provided by the heparin and the anti-platelet activity of the catalytically produced NO. It also endows the stents with the ability to simultaneously up-regulate α-smooth muscle actin (α-SMA) expression and to increase cyclic guanylate monophosphate (cGMP) synthesis of SMC, thereby significantly promoting their contractile phenotype and suppressing their proliferation. Importantly, this endothelium-biomimetic coating creates a favorable microenvironment for EC over SMC. These features impressively improve the antithrombogenicity, re-endothelialization and anti-restenosis of vascular stents in vivo.

A degradable triple temperature-, pH-, and redox-responsive drug system for cancer chemotherapy.

In order to achieve a precise therapeutic effect of cancer treatment and improve the utilization of drugs, a temperature-, pH-, and redox-responsive drug delivery system were synthesized. Methacrylic acid (MAA), poly(N-isopropylacrylamide) (PNIPAM), 2-hydroxyethylmethacrylate (HEMA), and N,N'-bis(acryloyl)cystamine (BACy), a disulfide bond contained cross-linker, were polymerized by a distillation-precipitation polymerization. Doxorubicin (DOX), an anti-cancer drug, can be loaded into the loose nanoparticle (NP) effectively. The prepared drug delivery remains stable during blood circulation and, when the vectors accumulated at tumor tissues, the pH-response of MMA and temperature-response of PNIPAM makes volume shrinkage of vectors which benefit the diffusion of vectors into tumor tissues. After being endocytosed into tumor cell, the disulfide bond that contained in the drug delivery can be cleaved by glutathione (GSH), causing the decomposition of NPs, and then release all of the drug. Under the influence of three trigger factors, the triple stimuli-responsive drug delivery vectors can realize tumor accumulation, tumor penetration and controlled drug release. Thus, the prepared multi-responsive NP is ideal drug carriers for developing novel drug delivery systems. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3203-3210, 2018.

Plant-inspired gallolamine catalytic surface chemistry for engineering an efficient nitric oxide generating coating.

A novel concept of generating therapeutic gas, nitric oxide (NO) via catalytic phenolic-amine "gallolamine" surface chemistry is developed. The concept is realized using plant polyphenol, gallic acid, and a glutathione peroxidase-like organoselenium compound cystamine or selenocystamine through one-step phenol-amine molecular assembling process. The resulting NO-generating coating with phenolic-cystamine or -selenocystamine framework showed the ability for long-term, steady and controllable range of NO release rates being unparalleled with any existing NO-releasing or NO-generating surface engineering toolkits. STATEMENT OF SIGNIFICANCE: Developing a facile and versatile strategy for a NO-generating coating with long-term, stable and adjustable NO release is of great interest for the application of blood-contacting materials and devices. Covalent immobilization of glutathione peroxidase (GPx)-like compound to generate NO from a material surface by exposure of endogenously existed S-nitrothiol (RSNO) is a popular strategy. However, it is generally involved in multi-step and complicated processes. Moreover, the amount of immobilized GPx-like compounds is limited by the density of introduced reactive functional groups on a surface. Herein, we propose a novel concept of catalytic plant-inspired gallolamine surface chemistry for material-independent NO-generating coatings. The concept is realized using plant polyphenol, gallic acid, and a GPx-like organoselenium compound cystamine or selenocystamine through one-step phenol-amine molecular assembling process. Without tedious multi-step synthesis, complicated surface treatments, and leakage of toxic chemicals, our unprecedentedly simple, histocompatible and biocompatible phenolic-cystamine or -selenocystamine framework demonstrated long-term, on-demand and facile dose controls of NO generated from the engineering surfaces. These unique features of such a NO-generating coating imparted a material with ability to impressively improve anti-thrombogenicity in vivo. This work constitutes the first report of an interfacial catalytic coating based on material-independent surface chemistry by plant polyphenols. This concept not only expands the application of material-independent surface chemistry in an interfacial catalytic area, but also can be a new platform for antithrombotic materials.

Mussel-inspired catalytic selenocystamine-dopamine coatings for long-term generation of therapeutic gas on cardiovascular stents.

The development of a nitric oxide (NO)-generating surface with long-term, stable and controllable NO release improves the therapeutic efficacy of cardiovascular stents. In this work, we developed a "one-pot" method inspired by mussel adhesive proteins for copolymerization of selenocystamine (SeCA) and dopamine (Dopa) to form a NO-generating coating on a 316 L stainless steel (SS) stent. This "one-pot" method is environmentally friendly and easy to popularize, with many advantages including simple manufacturing procedure, high stability and no involvement of organic solvents. Such SeCA/Dopa coatings also enabled us to develop a catalytic surface for local NO-generation by reaction of endogenously existing S-nitrothiol species from fresh blood. We found that the developed SeCA/Dopa coatings could release NO in a controllable and stable manner for more than 60 days. Additionally, the released NO significantly inhibited smooth muscle cell (SMC) proliferation and migration, as well as platelet activation and aggregation through the up-regulation of cyclic guanosine monophosphate synthesis. Moreover, such NO generation enhanced the adhesion, proliferation and migration of endothelial cells (ECs), and achieved rapid in vivo re-endothelialization, effectively reducing in-stent restenosis and neointimal hyperplasia. We envision that the SeCA/Dopa-coated 316 L SS stent could be a promising platform for treatment of cardiovascular diseases.

Facile Access to Multisensitive and Self-Healing Hydrogels with Reversible and Dynamic Boronic Ester and Disulfide Linkages.

Multifunctional and multiresponsive hydrogels have presented a promising platform to design and fabricate smart devices for application in a wide variety of fields. However, their preparations often involve multistep preparation of multiresponsive polymer precursors, tedious reactions to introduce functional groups or sophisticated molecular designs. In this work, a multifunctional boronic acid-based cross-linker bis(phenylboronic acid carbamoyl) cystamine (BPBAC) was readily prepared from inexpensive commercially available 3-carboxylphenylboronic acid (CPBA) and cystamine dihydrochloride, which has the ability to cross-link the cis-diols and catechol-containing hydrophilic polymers to form hydrogels. Due to the presence of the reversible and dynamic boronate ester and disulfide bonds, the obtained hydrogels were demonstrated to not only possess pH, glucose, and redox triresponsive features, but also have autonomic self-healing properties under ambient conditions. Moreover, we can modulate the rheological and mechanical properties by simply adjusting the BPBAC amount. The features, such as commercially available starting materials, easy-to-implement approach, and versatility in controlling cross-linking network and mechanical properties, make the strategy described here a promising platform for fabricating multifunctional and smart hydrogels.

Novel technique to determine the pKA of clonidine at prejunctional α2-adrenoceptors in cardiac and vascular sympathetic transmission.

Analytical pharmacology draws heavily on the concept of equilibrium of agonist and silent antagonist concentrations competing at a specific receptor site. This condition breaks down in nerve transmission when transmitter release is inhibited by prejunctional α2-adrenoceptors activated by an agonist such as clonidine. We have developed a method that allows the agonist dissociation constant KA of clonidine to be determined in a robust isolated right atrial assay of mouse, rat and guinea pig. By applying low numbers of field pulses 1-4 to prevent autoinhibitory feedback, clonidine shifted the nerve pulse stimulation-tachycardia curves to the right. These peak responses to field pulses were equated to responses to exogenous noradrenaline and the pKA determined by global fitting and display in the Clark plot. The pKA for clonidine ranged from 8.95 in the mouse, 7.8 in rat and 8.3 in guinea pig. The propranolol pKB was 8.87 in mouse and 8.91 in rat atria, reading very similarly to those values from ß-adrenoceptor agonist assays under equilibrium conditions. In mesenteric resistance arteries mounted in a myograph for electrical field stimulation, clonidine again inhibited contractions to field pulses in mouse arteries with a pKA of 7.12, but was inactive in rat arteries due to competing autoinhibitory feedback from nerve-released noradrenaline. In both species, prazosin inhibited the field pulses with a pKB of 9.08 in rat and 9.03 in mouse arteries. We conclude that pKB for antagonists and pKA for the prejunctional inhibitors of nerve transmission can be determined with this novel analytical approach.

Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.

Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency.

Poly(Amido Amine)s Containing Agmatine and Butanol Side Chains as Efficient Gene Carriers.

A new type of bioreducible poly(amido amine) copolymer is synthesized by the Michael addition polymerization of cystamine bisacrylamide (CBA) with 4-aminobutylguanidine (agmatine, AGM) and 4-aminobutanol (ABOL). Since the positively charged guanidinium groups of AGM and the hydroxybutyl groups of ABOL in the side chains have shown to improve the overall transfection efficiency of poly(amido amine)s, it is hypothesized that poly(CBA-ABOL/AGM) synthesized at the optimal ratio of both components would result in high transfection efficiency and minimal toxicity. In this study, a series of the poly(CBA-ABOL/AGM) copolymers is synthesized as gene carriers. The polymers are characterized and luciferase transfection efficiencies of the polymers in various cell lines are investigated to select the ideal ratio between AGM and ABOL. The poly(CBA-ABOL/AGM) containing 80% AGM and 20% ABOL has shown the best transfection efficiency with the lowest cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier.

Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection.

Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer-by-layer dip-coating of pCABOL with functional DNA is an easy and inexpensive method to introduce transfection capability to biomaterials of any nature and shape, which can be beneficially used in various biomedical and tissue engineering applications.

Controlling the reactivity of the Se-Se bond by the supramolecular chemistry of cucurbituril.

We describe a new strategy to control the reactivity of SeSe bond by using supramolecular chemistry of cucurbituril. We have demonstrated that selenocystamine (SeCy) and cucurbit[6]uril (CB[6]) can form a stable supramolecular complex (Ka =5.5×10(6) M(-1) ). Before complexation, the free SeSe bond in SeCy is rather sensitive to redox stimuli and gets disrupted quickly with addition of reductant or oxidant. However, after binding with CB[6], the SeSe bond becomes quite inert and hardly reacts with reductant or oxidant. One advantage of this supramolecular protection is that it can be applied in a wide pH range from weakly acidic to basic. Additionally, the supramolecular complex formed by SeCy and CB[6] can be reversibly dissociated simply with addition of Ba(2+) .

Construction and biofunctional evaluation of electrospun vascular graft loaded with selenocystamine for in situ catalytic generation of nitric oxide.

Construction and biofunctional evaluation of a novel vascular graft with in situ catalytic generation of nitric oxide were described in this paper. Poly α-lysine and poly (γ-glutamic acid) were deposited alternately onto the surface of an electrospun poly ε-caprolactone matrix via electrostatic layer-by-layer self-assembly, and then selenocystamine was loaded as a catalyst. Measurement of in vitro catalytic generation of nitric oxide demonstrated that this catalyst-loaded material could considerably accelerate the release of nitric oxide from S-nitrosoglutathione. A fibroblast proliferation assay showed that the material possessed satisfactory cellular compatibility. The catalyst-loaded material could inhibit the spread of smooth muscle cells in the presence of nitric oxide donors. In arteriovenous-shunt experiment, the catalyst-loaded graft exhibited good anti-thrombotic property where it could prevent acute thrombosis by decreasing the adhesion and activation of platelets and other blood cells. These data suggest a new method of building vascular grafts with improved hemocompatibility and biological functions.

Evidence for targeting thioredoxin reductases with ferrocenyl quinone methides. A possible molecular basis for the antiproliferative effect of hydroxyferrocifens on cancer cells.

Many anticancer compounds are strong inhibitors of thioredoxin reductases (TrxRs), selenoenzymes involved in cellular redox regulation. This study examined the effect of two hydroxyferrocifens (1, FcOH; 2, FcOHTAM) and of their corresponding quinone methides (QMs), 1-QM, and 2-QM, on these enzymes. In vitro, both QMs were more potent TrxR inhibitors (IC50 ≈ 2.5 µM) than the hydroxyferrocifens (IC50 ≈ 15 µM). This inhibition was due to a Michael addition of the penultimate selenocysteine residue of TrxRs to the QMs. In Jurkat cancer cells, both 2 and 2-QM inhibited TrxRs in the same proportion, leading to accumulation of oxidized forms of thioredoxin, while 1 and 1-QM were scarcely effective. This difference of behavior was ascribed to the competitive conversion of 1-QM to an inactive indene in protic medium. This set of experiments confirms for the first time the role played by ferrocenyl quinone methides on several biological targets and gives a molecular basis for these effects. It also highlights differences in the mechanisms of action of 1 and 2 in cancer cells.

Biodegradable large compound vesicles with controlled size prepared via the self-assembly of branched polymers in nanodroplet templates.

Generally, it is very difficult to control the size of large compound vesicles. Here, we introduce a novel method for the preparation of biodegradable large compound vesicles with controlled size and narrow size distribution by using aqueous nanodroplets as templates.

TLR4 and NLRP3 inflammasome activation in monocytes by N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD).

BACKGROUND: N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. OBJECTIVE: We investigated the effect of NPCMD on innate immune responses in monocytes. METHODS: CD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. RESULTS: NPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1ß in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1ß. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1ß secretion in treatment with NPCMD. Inhibition of IL-1ß secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. CONCLUSION: The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.

Nonchromatographic speciation of selenium in edible oils using dispersive liquid-liquid microextraction and electrothermal atomic absorption spectrometry.

A methodology for the nonchromatographic separation of the main selenium species present in edible oils is presented. Dispersive liquid-liquid microextraction is used to extract inorganic selenium (iSe), seleno-L-cystine (SeCys2), seleno-L-methionine (SeMet), and selenocystamine (SeCM) into a slightly acidic aqueous medium. The selenium total (tSe) content is measured in the extracts by electrothermal atomic absorption spectrometry. By repeating the microextraction stage using an ionic liquid instead of water, the sum of SeCys2, SeMet, and SeCM is obtained and iSe is calculated by difference. The detection limit is 0.03 ng of Se per gram of oil. The fractionation of the edible oils by solid phase extraction followed by dispersive liquid-liquid extraction and atomic absorption measurement also permits speciation of iSe to be carried out. Data for tSe and iSe levels of 15 samples of different origin are given.

Cystamine-terminated poly(beta-amino ester)s for siRNA delivery to human mesenchymal stem cells and enhancement of osteogenic differentiation.

Enhancing human mesenchymal stem cell (hMSC) differentiation via RNA interference (RNAi) could provide an effective way of controlling cell fate for tissue engineering, but a safe and effective delivery vehicle must first be developed. Here, we evaluated an array of synthetic end-modified poly(beta-amino ester) (PBAE)-based nanoparticles to optimize siRNA delivery into hMSCs. In general, cystamine-terminated polymers caused the most knockdown, with the best polymer achieving 91% knockdown 20 days post-transfection. Binding studies revealed that the cystamine-terminated polymer bound siRNA tightly at lower weight ratios of polymer to siRNA but then efficiently released siRNA upon exposure to a reducing environment, suggesting that this class of PBAEs can form tight initial interactions with its cargo and then cause efficient, environmentally-triggered release in the cytoplasm. Finally, we tested a functional application of this system by transfecting hMSCs with siRNA against an inhibitor of osteogenesis, B-cell lymphoma (Bcl)-like protein 2 (BCL2L2). This resulted in enhanced osteogenesis over 4 weeks as evidenced by Alizarin Red S staining and calcium quantification. The bioreducible PBAE/siRNA nanoparticles developed here can provide a means of safe and effective control of hMSC differentiation for a wide variety of applications.

Mechanism of putative neo-antigen formation from N-propionyl-4-S-cysteaminylphenol, a tyrosinase substrate, in melanoma models.

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.

N-propionyl-4-S-cysteaminylphenol induces apoptosis in B16F1 cells and mediates tumor-specific T-cell immune responses in a mouse melanoma model.

BACKGROUND: N-propionyl-4-S-cysteaminylphenol (NPr-4-S-CAP) is selectively incorporated into melanoma cells and degrades them. However, it remains unclear whether NPr-4-S-CAP can induce cell death associated with the induction of host immune responses and tumor suppression in vivo. OBJECTIVE: To examine the molecular mechanism of NPr-4-S-CAP-mediated cytotoxicity toward melanoma cells and to test whether NPr-4-S-CAP can suppress transplanted primary and secondary B16F1 melanomas. METHODS: Cytotoxicity and apoptosis of melanoma cells were assessed by cell counting, flow cytometry, and detection of reactive oxygen species (ROS) and apoptotic molecules. NPr-4-S-CAP-associated host immunity was studied using a B16F1 mouse melanoma model through the application of CD4- and CD8-specific antibodies and tetramer assay. RESULTS: NPr-4-S-CAP suppressed growth of pigmented melanoma cells associated with an increase of intracellular ROS, activation of caspase 3 and DNA fragmentation, suggesting that NPr-4-S-CAP mediated ROS production, eliciting apoptosis of melanoma cells. Growth of transplanted B16F1 melanomas was inhibited after the consecutive intratumoral injections of NPr-4-S-CAP, and the tumor growth after rechallenge of B16F1 was significantly suppressed in the treated mice. This suppression occurred when the treated mice were given the anti-CD4 antibody, but not the anti-CD8 antibody. Tetramer assay demonstrated increased TYRP-2-specific CD8(+) T cells in the lymph node and spleen cells prepared from NPr-4-S-CAP-treated B16F1-bearing mice. CONCLUSIONS: These suggest that NPr-4-S-CAP induces apoptosis in melanoma cells through ROS production and generates CD8(+) cell immunity resulting in the suppression of rechallenged B16F1 melanoma.