"Golgi-customized Trojan horse" nanodiamonds impair GLUT1 plasma membrane localization and inhibit tumor glycolysis.

Nutrient or energy deprivation, especially glucose restriction, is a promising anticancer therapeutic approach. However, establishing a precise and potent deprivation strategy remains a formidable task. The Golgi morphology is crucial in maintaining the function of transport proteins (such as GLUT1) driving glycolysis. Thus, in this study, we present a "Golgi-customized Trojan horse" based on tellurium loaded with apigenin (4',5,7-trihydroxyflavone) and human serum albumin, which was able to induce GLUT1 plasma membrane localization disturbance via Golgi dispersal leading to the inhibition of tumor glycolysis. Diamond-shaped delivery system can efficiently penetrate into cells as a gift like Trojan horse, which decomposes into tellurite induced by intrinsically high H2O2 and GSH levels. Consequently, tellurite acts as released warriors causing up to 3.8-fold increase in Golgi apparatus area due to the down-regulation of GOLPH3. Further, this affects GLUT1 membrane localization and glucose transport disturbance. Simultaneously, apigenin hinders ongoing glycolysis and causes significant decrease in ATP level. Collectively, our "Golgi-customized Trojan horse" demonstrates a potent antitumor activity because of its capability to deprive energy resources of cancer cells. This study not only expands the applications of tellurium-based nanomaterials in the biomedicine but also provides insights into glycolysis restriction for anticancer therapy.

Remodeling of the liver fibrosis microenvironment based on nilotinib-loaded multicatalytic nanozymes with boosted antifibrogenic activity.

Liver fibrosis is a reversible pathological process caused by chronic liver damage and a major risk factor for hepatocellular carcinoma (HCC). Hepatic stellate cell (HSC) activation is considered the main target for liver fibrosis therapy. However, the efficiency of this strategy is limited due to the complex microenvironment of liver fibrosis, including excessive extracellular matrix (ECM) deposition and hypoxia-induced imbalanced ECM metabolism. Herein, nilotinib (NIL)-loaded hyaluronic acid (HA)-coated Ag@Pt nanotriangular nanozymes (APNH NTs) were developed to inhibit HSCs activation and remodel the microenvironment of liver fibrosis. APNH NTs efficiently eliminated intrahepatic reactive oxygen species (ROS) due to their inherent superoxide dismutase (SOD) and catalase (CAT) activities, thereby downregulating the expression of NADPH oxidase-4 (NOX-4) and inhibiting HSCs activation. Simultaneously, the oxygen produced by the APNH NTs further alleviated the hypoxic microenvironment. Importantly, the released NIL promoted collagen depletion by suppressing the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), thus synergistically remodeling the microenvironment of liver fibrosis. Notably, an in vivo study in CCl4-induced mice revealed that APNH NTs exhibited significant antifibrogenic effects without obvious long-term toxicity. Taken together, the data from this work suggest that treatment with the synthesized APNH NTs provides an enlightening strategy for remodeling the microenvironment of liver fibrosis with boosted antifibrogenic activity.

Mapping of MeLiM melanoma combining ICP-MS and MALDI-MSI methods.

Here we developed a powerful tool for comprehensive data collection and mapping of molecular and elemental signatures in the Melanoma-bearing Libechov Minipig (MeLiM) model. The combination of different mass spectrometric methods allowed for detail investigation of specific melanoma markers and elements and their spatial distribution in tissue sections. MALDI-MSI combined with HPLC-MS/MS analyses resulted in identification of seven specific proteins, S100A12, CD163, MMP-2, galectin-1, tenascin, resistin and PCNA that were presented in the melanoma signatures. Furthermore, the ICP-MS method allowed for spatial detection of zinc, calcium, copper, and iron elements linked with the allocation of the specific binding proteins.

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells.

BACKGROUND: Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS: The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% ß-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION: This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

Metallothionein isoforms as double agents - Their roles in carcinogenesis, cancer progression and chemoresistance.

Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.

Prostate cancer-specific hallmarks of amino acids metabolism: Towards a paradigm of precision medicine.

So far multiple differences in prostate cancer-specific amino acids metabolism have been discovered. Moreover, attempts to utilize these alterations for prostate cancer diagnosis and treatment have been made. The prostate cancer metabolism and biosynthesis of amino acids are particularly focused on anaplerosis more than on energy production. Other crucial requirements on amino acids pool come from the serine, one­carbon cycle, glycine synthesis pathway and folate metabolism forming major sources of interproducts for synthesis of nucleobases necessary for rapidly proliferating cells. Considering the lack of some amino acids biosynthetic pathways and/or their extraordinary importance for prostate cancer cells, there is a widespread potential for targeted therapeutic applications with no effect on non-malignant cells. This review summarizes the up-to-date knowledge of the importance of amino acids for prostate cancer pathogenesis with a special emphasis on potential applications of metabolic variabilities in the new oncologic paradigm of precision medicine.

Sarcosine is a prostate epigenetic modifier that elicits aberrant methylation patterns through the SAMe-Dnmts axis.

DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine-induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.

Mass spectrometric imaging of cysteine rich proteins in human skin.

Looking insight pathological processes, metallothioneins (MTs) are considered to be potential biomarkers for monitoring of a development of various types of diseases, such as cancer. The early identification of the MTs in biological tissues could be important tool for the estimation of appropriate clinical therapy. Therefore, here we investigated the application of matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) together with immunohistochemical analyses (IHC) using MT-1/2 antibody for MT detection in formalin-fixed paraffin-embedded (FFPE) biopsy specimens of human skin. Principal component analyses revealed differences in the peptide/protein profiles separating healthy skin from the carcinoma specimens. Statistically significant ion peaks at m/z 6038, 6300, 6676, and 7026 were more frequently detected in squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Using IHC, we found that MT-1/2 was significantly higher in SCC and melanoma compared to healthy skin. Surprisingly, significantly low levels of MT-1/2 were found in BCC. On one side, the results indicate important role of MTs in melanoma occurrence and progression, as on the second side, there are hidden processes associated with MTs based on differences of the occurrence of the MS peaks, which could be associated with cycling of MTs isoforms.

Current Trends in Detection of Histamine in Food and Beverages.

Histamine is a heterocyclic amine formed by decarboxylation of the amino acid l-histidine. It is involved in the local regulation of physiological processes but also can occur exogenously in the food supply. Histamine is toxic at high intakes; therefore, determination of the histamine level in food is an important aspect of food safety. This article will review the current understanding of physiological functions of endogenous and ingested histamine with a particular focus placed on existing and emerging technologies for histamine quantification in food. Methods reported in this article are sequentially arranged and provide a brief overview of analytical methods reported, including those based on nanotechnologies.

The application of capillary electrophoresis, mass spectrometry and Brdicka reaction in human and rabbit metallothioneins analysis.

BACKGROUND: Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells. OBJECTIVES: The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared. MATERIAL AND METHODS: Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. RESULTS: In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT. CONCLUSIONS: The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.

Preliminary analysis of the interactions between CdTe quantum dots and human metallothionein.

Metallothionein (MT) plays the important role in the detoxification of heavy metals, protection against oxidative compounds and as a prognostic marker in the development of tumors. It is important to find selective, stable and sensitive tools and probes to evaluate the presence of MT in biological fluids or tissues. QDs linked with ligands such as peptides or small molecules are a promising tool for selective, fast, and sensitive tagging and imaging in medicine. In previous findings, the authors proved the possibility of interaction with QDs (particularly with CdTe) and analyzed the stability of the formed complexes between CdTe and MT during incubation over time. Following that, an initial analysis of the interactions between CdTe quantum dots (QDs) and human metallothionein (MT) was performed. Complexes of mercaptosuccinic acid-covered CdTe QDs + MT were investigated using fluorescence intensity changes along a timeline, quenching analysis, stability interpretation based on zeta potential, and quenching intensity. Based on the preliminary results, it appears as though the possible interactions depend on the size of the CdTe QDs. Additionally, the formation of complexes between CdTe and human MT likely depends mostly on structural changes and conformational reorganization rather than on electrostatic interactions. Both types of interactions are responsible for complex creation and stabilization.

Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin.

Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

Improving cytocompatibility of CdTe quantum dots by Schiff-base-coordinated lanthanides surface doping.

BACKGROUND: Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. RESULTS: Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9-58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target-human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. CONCLUSION: For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.

Short-sweep capillary electrophoresis with a selective zinc fluorescence imaging reagent FluoZin-3 for determination of free and metalothionein-2a-bound Zn2+ ions.

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn2+ and Cd2+ as complexes with highly selective and sensitive fluorescent probe FluoZin-3. The ShortSweepCE-LIF method, established in this work, can be used for examining competitive Zn2+ and Cd2+ binding properties of metalloproteins or peptides. The parameters including background electrolyte composition, injection pressure and time as well as separation voltage were investigated. Under the optimized conditions, 80 mM HEPES, pH 7.4, with 1.5 µM FluoZin-3 was used as an electrolyte, hydrodynamic injection was performed at 50 mbar for 5 s, and separation voltage of 25 kV. Limits of detection for Zn2+ and Cd2+ were 4 and 125 nM, respectively. The developed method was demonstrated in a study of interactions between metalothionein-2a isoform and metal ions Zn2+, Co2+ and Cd2+. It was found that FluoZin-3 was able to extract a single Zn2+ ion, while added Co2+ (in surplus) extracted only 2.4 Zn2+ ions, and Cd2+ extracted all 7 Zn2+ ions present in the metalothionein molecule.

Comparative gene expression profiling of human metallothionein-3 up-regulation in neuroblastoma cells and its impact on susceptibility to cisplatin.

Human metallothionein-3 (hMT-3), also known as growth inhibitory factor, is predominantly expressed in the central nervous system. hMT-3 is presumed to participate in the processes of heavy metal detoxification, regulation of metabolism and protection against oxidative damage of free radicals in the central nervous system; thus, it could play important neuromodulatory and neuroprotective roles. However, the primary functions of hMT-3 and the mechanism underlying its multiple functions in neuroblastoma have not been elucidated so far. First, we confirmed relatively high expression of hMT-3 encoding mRNA in biopsies (n = 23) from high-risk neuroblastoma subjects. Therefore, we focused on investigation of the impact of hMT-3 up-regulation in N-Myc amplifying neuroblastoma cells. The differentially up-regulated genes involved in biological pathways related to cellular senescence and cell cycle were identified using electrochemical microarray with consequent bioinformatic processing. Further, as experimental verification of microarray data, the cytotoxicity of the cisplatin (CDDP) was examined in hMT-3 and mock cells by MTT and clonogenic assays. Overall, our data strongly suggest that up-regulation of hMT-3 positively correlates with the genes involved in oncogene-induced senescence (CDKN2B and ANAPC5) or apoptosis (CASP4). Moreover, we identified a significant increase in chemoresistance to cisplatin (CDDP) due to hMT-3 up-regulation (24IC50: 7.5 vs. 19.8 µg/ml), indicating its multipurpose biological significance.

An insight into the complex roles of metallothioneins in malignant diseases with emphasis on (sub)isoforms/isoforms and epigenetics phenomena.

Metallothioneins (MTs) belong to a group of small cysteine-rich proteins that are ubiquitous throughout all kingdoms. The main function of MTs is scavenging of free radicals and detoxification and homeostating of heavy metals. In humans, 16 genes localized on chromosome 16 have been identified to encode four MT isoforms labelled by numbers (MT-1-MT-4). MT-2, MT-3 and MT-4 proteins are encoded by a single gene. MT-1 comprises many (sub)isoforms. The known active MT-1 genes are MT-1A, -1B, -1E, -1F, -1G, -1H, -1M and -1X. The rest of the MT-1 genes (MT-1C, -1D, -1I, -1J and -1L) are pseudogenes. The expression and localization of individual MT (sub)isoforms and pseudogenes vary at intra-cellular level and in individual tissues. Changes in MT expression are associated with the process of carcinogenesis of various types of human malignancies, or with a more aggressive phenotype and therapeutic resistance. Hence, MT (sub)isoform profiling status could be utilized for diagnostics and therapy of tumour diseases. This review aims on a comprehensive summary of methods for analysis of MTs at (sub)isoforms levels, their expression in single tumour diseases and strategies how this knowledge can be utilized in anticancer therapy.

MALDI MSI of MeLiM melanoma: Searching for differences in protein profiles.

BACKGROUND: Treatment of advanced cutaneous melanoma remains challenging, and new data on melanoma biology are required. The most widely accepted criteria for the prognostic evaluation of melanoma are histopathological and clinical parameters, and the identification of additional tumor markers is thus of paramount importance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI), an important tool in cancer research, is useful for unraveling the molecular profile of melanoma. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we used the melanoma-bearing Libechov minipig (MeLiM), a unique animal model that allows observation of the complete spontaneous regression of invasive cutaneous melanoma, to investigate i) the differences between melanoma and healthy skin protein profiles and ii) the proteins potentially involved in spontaneous regression. The MeLiM tissues were cryosected, histologically characterized, analyzed by MALDI MSI, and immunohistologically stained. Multivariate statistical analyses of the MALDI MSI data revealed ten relevant m/z ions, of which the expression levels varied significantly among the studied MeLiM tissues. These ion peaks were used to create mass ion images/maps and visualize the differences between tumor and healthy skin specimens, as well as among histologically characterized tissue regions. CONCLUSIONS/SIGNIFICANCE: Protein profiles comprising ten statistically significant mass ion peaks useful for differentiating cutaneous melanoma and healthy skin tissues were determined. Peaks at m/z 3044, 6011, 6140 and 10180 were overexpressed in melanoma compared with healthy skin tissue. More specifically, m/z 6140 was expressed at significantly (p < 0.05) higher levels in normally growing melanoma regions than in regions with early and late spontaneous regression. This study demonstrates the clinical utility of MALDI MSI for the analysis of tissue cryosections at a molecular level.

Kinetic analysis of human metallothionein and CdTe quantum dot complexes using fluorescence and voltammetry techniques.

Thanks to quantum dots' (QDs) properties, they can be used as selective and sensitive biomarkers in molecular imaging. In a previous paper, we confirmed the possibility of interaction between mercaptosuccinic acid-capped cadmium telluride QDs (MSA-CdTe) and human metallothionein (MT). The aim of this study was to expand on our previous research with an evaluation of the stability of the formed complexes between human MT and four CdTe compounds of the following sizes: 3.4nm (blue QDs), 3.8nm (green QDs), 4.5nm (yellow QDs), and 5.2nm (red QDs). Complexes were evaluated over time using fluorescence intensity and differential pulse voltammetry. Differences between the voltammograms obtained for standard solutions and for CdTe+MT show that complexes were formed. An increase in fluorescence intensity was observed for blue (Δ%≈40 for t=1→120min) and red (Δ%≈30 for t=1→120min) CdTe-MT complexes than CdTe alone, whereas green and yellow CdTe-MT complexes had a lower fluorescence intensity than CdTe alone. A stronger time dependence of the mercaptosuccinic acid (MSA) peak height on the timeline and differences in the MSA peak shape (in CdTe, and CdTe+MT complexes) were also observed by voltammetry. Authors noticed a decrease in the Cat2 signal of the red and green CdTe+MT complexes at the time of conjugation. Our results reveal that the size of QDs has an impact on the interaction between CdTe and human MT, as well as on the stability of complexes formed during these interactions. The bioconjugates' stability was also found to depend on the time of interaction.