Search

Rationale: The binding ratio of metal complexes with cysteinyl thiols in proteins plays an important role in deciphering the mechanisms of action of therapeutic metal complexes, but its analysis is still a significant challenge. In this work, a quantitative mass spectrometry method is developed to determine the binding ratio of metal-based anticancer complexes with cysteines in human copper chaperone protein Atox1., Methods: A novel strategy based on a thiol-specific stable isotopic labelling reagent was developed to determine the binding ratio of metal-based anticancer complexes, namely cisplatin and organometallic ruthenium complex [(η 6 -biphenyl)RuCl(en)]PF 6 (en = ethylenediamine), with the cysteinyl residues of Atox1., Results: Both cisplatin and the ruthenium complex were reactive not only to Cys15 and/or Cys18, the copper(I) binding site of Atox1, but also to Cys44. The binding ratios of the ruthenium complex with the cysteinyl residues were much higher than those of cisplatin. However, the addition of copper(I) could markedly increase the binding ratios of cysteinyl residues of Atox1 with cisplatin, but not with the ruthenium complex., Conclusions: This strategy can not only precisely determine the binding ratios of metal complexes to protein thiols, but also be helpful in distinguishing thiol-binding sites from other binding sites of metal complexes in proteins. We expect wide application of this method to the research of covalent/coordinative interactions between metal complexes and protein thiols., (© 2019 John Wiley & Sons, Ltd.)

Strophanthidin (SPTD), one of the cardiac glycosides, is refined from traditional Chinese medicines such as Semen Lepidii and Antiaris toxicaria, and was initially used for the treatment of heart failure disease in clinic. Recently, SPTD has been shown to be a potential anticancer agent, but the underlying mechanism of action is poorly understood. Herein, we explored the molecular mechanism by which SPTD exerts anticancer effects in A549 human lung adenocarcinoma cells by means of mass spectrometry-based quantitative proteomics in combination with bioinformatics analysis. We revealed that SPTD promoted the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2, or DR5) in A549 cells to activate caspase 3/6/8, in particular caspase 3. Consequently, the activated caspases elevated the expression level of apoptotic chromatin condensation inducer in the nucleus (ACIN1) and prelamin-A/C (LMNA), ultimately inducing apoptosis via cooperation with the SPTD-induced overexpressed barrier-to-autointegration factor 1 (Banf1). Moreover, the SPTD-induced DEPs interacted with each other to downregulate the p38 MAPK/ERK signaling, contributing to the SPTD inhibition of the growth of A549 cells. Additionally, the downregulation of collagen COL1A5 by SPTD was another anticancer benefit of SPTD through the modulation of the cell microenvironment. [ABSTRACT FROM AUTHOR]

Four trinucleotides 5′-ATA-3′ (I), 5′-ATC-3′ (II), 5′-CTA-3′ (III) and 5′-CTC-3′ (IV) were introduced to interact with a diazido-based photoactivatable anticancer prodrug trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (py = pyridine; 1) upon light irradiation. Using electrospray ionization mass spectrometry (ESI-MS), we aimed to investigate the possibility of 1,3-intrastrand crosslinks at adenine and/or cytosine in the trinucleotides via the bi-functional trans-[PtII(py)2]2+ species generated by photodecomposition of complex 1. The primary mass spectrometry results showed that although mono- and di-platinated trinucleotides bound by mono-functional trans-[PtII(N3)(py)2]+ species were the major platinated adducts, comparable amounts of bifunctional trans-[PtII(py)2]2+-bound trinucleotides were also observed. Further tandem mass spectrometry of the trans-[PtII(py)2]2+-bound trinucleotides showed the formation of 1,3-crosslinks between adenine–adenine, adenine–cytosine and cytosine–cytosine bases in the trinucleotides. The formation of such unique structures is not only distinct from the action modes of cisplatin with DNA but also an important complement to the acknowledged 1,3-GNG intrastrand crosslink by trans-Pt species, which may support the promising and distinct anticancer activities of such photoactivatable diazido Pt(IV) anticancer prodrugs and deserve further studies. [ABSTRACT FROM AUTHOR]

The interaction of a photoactivatable diazidodihydroxido Pt(IV) prodrug, trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1), with a hexamer straight human telomeric DNA unit sequence (5′-T1T2A3G4G5G6-3′, I) upon light irradiation was investigated by electrospray ionization mass spectroscopy (ESI-MS). In the primary mass spectrum, two major mono-platinated I adducts with the bound Pt moieties, trans-[PtII(N3)(py)2]+ (1′) and trans-[PtII(py)2]2+ (1′′), respectively, were detected. It is rare to observe such high abundance and nearly equal intensity platinated DNA adducts formed by these two PtII species because 1′ is usually the only major reduced Pt(II) species produced by the photodecomposition of complex 1 in the presence of DNA while 1′′ was rarely detected as the major reduced PtII species reported previously. Subsequent tandem mass spectrometric analysis by collision-induced dissociation (CID) showed that in the former adduct {I + 1′}2+, G6 and A3 were the platination sites. While in the latter adduct {I + 1′′}2+, a potential intrastrand crosslink was speculated after G4 and G6 sites were identified. Additionally, other minor platinated adducts like di-platinated I adduct by 1′ with platination sites at G4 and G6 and mono-platinated I adducts containing base oxidation were also detected by mass spectrometry. Due to the rich guanines and their sensitivity to oxidation, the oxidation induced by 1 most probably occurred at guanine. The oxidation adducts were proposed as 8-hydroxyl guanine, spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 5-guanidinohydantoin (Gh), and/or dehydroguanidinohydantoin (DGh) referring to previous reports. The obtained results provide useful chemical information about the photoreaction between photoactivatable Pt(IV) anticancer prodrugs and human telomeric DNA. Such special damages of Pt(IV) prodrugs on human telomeric DNA implicate its active role in the mechanism of Pt(IV) prodrugs and further support the unique sequence-dependent photointeraction profile of complex 1 reacting with DNA. [ABSTRACT FROM AUTHOR]

We report here the development of clickable and highly near‐infrared (NIR) fluorescent lanthanide (Ln) complexes for bioorthogonal labeling of biomolecules. These azide‐ or alkyne‐functionalized Ln complexes are hydrophilic and fluorogenic, exhibiting a strong increase of NIR fluorescence upon conjugation with biomolecules. Metabolic labeling of biomolecules with azide or alkyne, followed by click labeling with the Ln complexes, enables NIR fluorescence (NIRF) imaging of DNA, RNA, proteins, and glycans in cells. Furthermore, multicolor imaging is performed by combining click‐labeling with the Ln complexes and immunostaining. In addition, the Ln complexes is compatible with click‐expansion microscopy (click‐ExM), which enables high‐resolution NIRF imaging of cellular glycoproteins. Finally, the Ln complexes can be used for time‐of‐flight secondary‐ion mass spectrometry (ToF‐SIMS) imaging, thus achieving the first example of dual‐modal imaging combining NIRF and SIMS microscopies. [ABSTRACT FROM AUTHOR]

Correction for 'Open-flow microperfusion combined with mass spectrometry for in vivo liver lipidomic analysis' by Tuo Li et al., Analyst, 2021, 146, 1915–1923, https://doi.org/10.1039/D0AN02189J. [ABSTRACT FROM AUTHOR]

At present, conventional microdialysis (MD) techniques cannot efficiently sample lipids in vivo, possibly due to the high mass transfer resistance and/or the serious adsorption of lipids onto the semi-permeable membrane of a MD probe. The in vivo monitoring of lipids could be of great significance for the study of disease development and mechanisms. In this work, an open-flow microperfusion (OFM) probe was fabricated, and the conditions for sampling lipids via OFM were optimized. Using OFM, the recovery of lipid standards was improved to more than 34.7%. OFM is used for the in vivo sampling of lipids in mouse liver tissue with fibrosis, and it is then combined with mass spectrometry (MS) to perform lipidomic analysis. 156 kinds of lipids were identified in the dialysate collected via OFM, and it was found that the phospholipid levels, including PC, PE, and SM, were significantly higher in a liver suffering from fibrosis. For the first time, OFM combined with MS to sample and analyze lipids has provided a promising platform for in vivo lipidomic studies. [ABSTRACT FROM AUTHOR]

Platinum based anticancer agents are widely applied in clinic and their major target is believed to be DNA. Herein, the interaction of a photoactivatable diazido Pt(IV) anticancer prodrug trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1) with a 15-mer single-G-containing oligodeoxynucleotide (ODN I: 5′-CT2CTCTTG8T9CT11TCTC-3′) was investigated by mass spectrometric methods. Up to penta-platinated ODN I adducts were identified from primary mass spectra while the mono- and di-platinated adducts had the highest intensity. Fragmentation of mono-, di- and tri-platinated I adducts in tandem MS revealed that T2, G8, T11 and T9 are binding sites. No cytosine sites were identified which may be due to the facile loss of Pt adducts from cytosine during CID. The intensity of {Pt(py)2}-bound adducts was comparable to that of {Pt(N3)(py)2}-bound adducts, indicating that the photo-reduction pathway of complex 1 from Pt(IV) to Pt(II) through two one-electron donations from two azides was substantial. Moreover, no transformation of N3 to NH3 on the {Pt(N3)(py)2}-bound adducts was observed, whereas it is very popular during the reactions of complexes with short ODNs or mono-nucleotides. The oxidation on I induced by the reactive oxygen species (ROS) formed by the photodecomposition of complex 1 was significant, and the oxidation of G8 to 8-hydroxyguanine (8-OH-G), spiroiminodihydantoin (Sp) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) was discovered. These results unambiguously revealed a sequence-length-dependent photochemical reactivity of complex 1 when it interacted with different ODNs, providing deeper understanding in the reactivity of photoactivatable diazido anticancer Pt(IV) prodrugs to DNA. [ABSTRACT FROM AUTHOR]

Rationale: Human telomeric DNA is reported to be a potential target for anticancer organometallic ruthenium(II) complexes, however, the interaction sites were not clearly discriminated and identified. Methods: In the current study, tandem mass spectrometry (MS/MS) using collision‐induced dissociation (CID) was firstly introduced to identify the interaction sites of an organometallic ruthenium(II) complex [(η6‐biphenyl)Ru(en)Cl][PF6] (1; en = ethylenediamine) with 5′‐T1T2A3G4G5G6‐3′ (I), the repeating unit of human telomeric DNA, in both positive‐ and negative‐ion mode at a low reaction molar ratio (1/I = 0.2) which was applied to preserve the site selectivity. Results: Mass spectrometric results showed that mono‐ruthenated I was the main product under the conditions. In positive‐ion mode, MS/MS results indicated that ruthenium complex 1 binds to T2 or G6 in strand I. However, in negative‐ion mode, no efficient information was obtained for exact identification of ruthenation sites which may be attributed to losses of fragment ions due to charge neutralization by the coordination of the positively charged ruthenium complex to the short MS/MS fragments. Conclusions: This is the first report of using top‐down MS to characterize the interactions of organometallic ruthenium(II) complexes and human telomeric DNA. Thymine can be thermodynamically competitive with guanine for binding to ruthenium complexes even at low reaction molar ratio, which inspired us to explore in greater depth the significance of thymine binding. [ABSTRACT FROM AUTHOR]

An addressable single cell imaging strategy combining ToF-SIMS and confocal fluorescence microscopy imaging has been developed, and sucessfully applied to visualize the subcellular distribution of an organoruthenium anticancer complex, [(η6-benzene)Ru(N,N-L)Cl]+ (1; L: 4-anilinoquinazoline ligand), showing its accumulation in both cell membrane and nuclei, and verifying its dual-targeting feature. [ABSTRACT FROM AUTHOR]

Rationale Cox17 is a key copper chaperone protein responsible for delivery of cuprous ions to mitochondria and has been demonstrated to be involved in the anticancer action of cisplatin. However, the binding sites of the drug to the protein have not yet been directly identified. Methods The recombinant protein apo-Cox172s-s, the functional state of Cox17 transferring Cu(I), was reacted with an excess of cisplatin to produce platinated Cox17 adducts, of which the platination sites were identified by high-resolution Fourier transform ion cyclotron tandem mass spectrometry (FT-ICR-MS/MS) through electron capture dissociation (ECD). Results Primary FT-ICR-MS showed that mono-platinated Cox17 adducts were the main products, and top-down MS/MS results indicated that cisplatin bound to the Cys26 or Cys27 residue which is the binding site of cuprous ions in apo-Cox172s-s. Conclusions This is the first report for identification of the main binding sites of cisplatin to Cox17 by top-down high-resolution mass spectrometry, providing direct evidence for the competitive coordination with Cox17 of cisplatin and cuprous ions. These findings will also be helpful to understand further how Cox17 facilitates cisplatin accumulation in mitochondria, and how cisplatin disturbs the transportation of cuprous ions. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Keywords: Bioorthogonal Labeling; Lanthanide; Mass Spectrometry Imaging; NIR Fluorescence Imaging EN Bioorthogonal Labeling Lanthanide Mass Spectrometry Imaging NIR Fluorescence Imaging 1 1 1 10/21/22 20221024 NES 221024 B Klickbare Lanthanid-Molekülsonden b sind im Titelbild als Pfeilspitzen dargestellt, und die beiden Bildgebungsverfahren NIRF und SIMS werden von zwei Adlern repräsentiert, deren Silhouetten sich aus Ausschnitten von NIR-Fluoreszenz- und Massenspektrometrie-Abbildugen zusammensetzen. Wie Yao Zhao, Xing Chen, Jun-Long Zhang et al. in ihrem Forschungsartikel (e202208707) berichten, wurde mit diesen klickbaren Lanthanid-Molekülsonden eine duale NIRF-SIMS-Bildgebung erreicht. Bioorthogonal Labeling, Lanthanide, NIR Fluorescence Imaging, Mass Spectrometry Imaging. [Extracted from the article]

We here report the identification of the binding sites of an organometallic ruthenium anticancer complex [(η6-biphenyl)Ru(en)Cl]+ (1) to single-stranded oligodeoxynucleotides (ODNs) 5′-CCCA4G5C6CC-3′ (I) and 5′-CCC3G4A5CCC-3′ (II) by mass spectrometry. The MS analysis of exonuclease ladders demonstrated that the 5′-exonuclease bovine spleen phosphodiesterase digestion of I and II mono-ruthenated by complex 1 was arrested solely at A4 and partially at C3 and G4, respectively, and that the 3′-exonuclease snake venom phosphodiesterase digestion of the ruthenated ODNs was arrested solely at G5 and G4, respectively, due to the ruthenation. These results did not allow unambiguous identification of ruthenation sites on the metallated ODNs. In contrast, tandem mass spectrometry analysis with CID fragmentation of the mono-ruthenated ODNs provided sequential and complementary [ai− B]/wi fragments, leading to unambiguous identification of G5 in I and G4 in II as the ruthenation sites on the ODN adducts, which is in line with the high selectivity of the complex towards guanine base as reported previously. These findings suggest that caution should be raised with regard to the identification of the binding sites of metal complexes, in particular complexes with bulky ligands, like biphenyl in complex 1, to DNA by MS analysis of exonuclease ladders of the metallated adducts, because the bulky ligands may adopt such an orientation that they block the exonuclease cleavage of the 5′- or 3′-side phosphodiester bonds adjacent to the binding sites, leading to digestion stalling at the nucleotides before the binding sites. [ABSTRACT FROM AUTHOR]

Reactions of the anticancer complex [(η6-bip)Ru(en)Cl]+ (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione (γ-l-Glu-l-Cys-Gly; GSH), the abundant intracellular thiol, in aqueous solution give rise to two ruthenium cluster complexes, which could not be identified by electrospray mass spectrometry (ESI-MS) using a quadrupole mass analyzer. Here we use Fourier transform ion cyclotron mass spectrometry (nanoLC-FT-ICR MS) to identify the clusters separated by nanoscale liquid chromatography as the tetranuclear complex [{(η6-bip)Ru(GSO2)}4]2− (2) and dinuclear complex [{(η6-bip)Ru(GSO2)2}2]8− (3) containing glutathione sulfinate (GSO2) ligands. Use of 18OH2 showed that oxygen from water can readily be incorporated into the oxidized glutathione ligands. These data illustrate the power of high-resolution MS for identifying highly charged multinuclear complexes and elucidating novel reaction pathways for metallodrugs, including ligand-based redox reactions. [Copyright &y& Elsevier]

Baicalin is a major active ingredient of traditional Chinese medicine Scutellaria baicalensis, and has been shown to have antiviral, anti-inflammatory, and antitumor activities. However, the protein targets of baicalin have remained unclear. Herein, a chemical proteomics strategy was developed by combining baicalin-functionalized magnetic nanoparticles (BCL-N3@MNPs) and quantitative mass spectrometry to identify the target proteins of baicalin. Bioinformatics analysis with the use of Gene Ontology, STRING and Ingenuity Pathway Analysis, was performed to annotate the biological functions and the associated signaling pathways of the baicalin targeting proteins. Fourteen proteins in human embryonic kidney cells were identified to interact with baicalin with various binding affinities. Bioinformatics analysis revealed these proteins are mainly ATP-binding and/or ATPase activity proteins, such as CKB, HSP86, HSP70-1, HSP90, ATPSF1β and ACTG1, and highly associated with the regulation of the role of PKR in interferon induction and the antiviral response signaling pathway (P = 10−6), PI3K/AKT signaling pathway (P = 10−5) and eNOS signaling pathway (P = 10−4). The results show that baicalin exerts multiply pharmacological functions, such as antiviral, anti-inflammatory, antitumor, and antioxidant functions, through regulating the PKR and PI3K/AKT/eNOS signaling pathways by targeting ATP-binding and ATPase activity proteins. These findings provide a fundamental insight into further studies on the mechanism of action of baicalin. [ABSTRACT FROM AUTHOR]

The in situ molecular characterization of reaction intermediates and products at electrode-electrolyte interfaces is central to mechanistic studies of complex electrochemical processes, yet a great challenge. The coupling of electrochemistry (EC) and mass spectrometry (MS) has seen rapid development and found broad applicability in tackling challenges in analytical and bioanalytical chemistry. However, few truly in situ and real-time EC-MS studies have been reported at electrode-electrolyte interfaces. An innovative EC-MS coupling method named in situ liquid secondary ion mass spectrometry (SIMS) was recently developed by combining SIMS with a vacuum compatible microfluidic electrochemical device. Using this novel capability, we report the first in situ elucidation of the electro-oxidation mechanism of a biologically significant organic compound, ascorbic acid (AA), at the electrode-electrolyte interface. The short-lived radical intermediate was successfully captured, which had not been detected directly before. Moreover, we demonstrated the power of this new technique in real-time monitoring of the formation and dynamic evolution of electrical double layers at the electrode-electrolyte interface. This work suggests further promising applications of in situ liquid SIMS in studying more complex chemical and biological events at the electrode-electrolyte interface. [ABSTRACT FROM AUTHOR]

Electrospray ionization mass spectrometry (ESI-MS) has been widely used to identify binding sites of metal complexes to proteins. However, the MS quantification of the metal–protein coordination remains a challenge. We have recently demonstrated by ESI-MS analysis that organometallic ruthenium complexes [(η 6 -arene)Ru(en)Cl] + (arene = p -cymene ( 1 ), biphenyl ( 2 ) or 9,10-dihydrophenanthrene ( 3 ); en = ethylenediamine) bound to human glutathione- S -transferase π (GSTπ) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GSTπ dimer, showing inhibitory potency against the enzyme (J. Inorg. Biochem., 128 (2013) 77–84). Herein, we developed a mass spectrometric method to quantify the binding stoichiometry of the three complexes to GSTπ. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GSTπ complexes and the respective light-labelled peptides in the tryptic digest of equimolar GSTπ were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GSTπ with its substrate GSH significantly reduced the bindings of the ruthenium complexes at Met92 and Cys102, but had little impact on the bindings at Cys15 and Cys48. As the inhibitory activities of the ruthenium complexes against GSTπ are similar to those against GSTπ in complexation with GSH, these results suggest that the inhibition of the ruthenium complexes on GSTπ is attributed to the ruthenation at Cys15 and Cys48. The present work provides not only insights into the understanding on the inhibitory mechanism of ruthenium complexes GSTπ, but also a general method for quantitative characterization of metal–protein interactions. [ABSTRACT FROM AUTHOR]

Abstract: Mass spectrometric quantification of phosphopeptides is a challenge due to the ion suppression effect of highly abundant non-phosphorylated peptides in complex samples such as serum. Several strategies for relative quantification of serum phosphopeptides based on MS have been developed, but the power of relative quantities was limited when making direct comparisons between two groups of samples or acting as a clinical examination index. Herein, we describe an MS absolute quantification strategy combined with Titania Coated Magnetic Hollow Mesoporous Silica Microspheres (TiO2/MHMSM) enrichment and stable isotopic acetyl labeling for phosphopeptides in human serum. Four endogenous serum phosphopeptides generated by degradation of fibrinogen were identified by LC–ESI–MS/MS following TiO2/MHMSM enrichment. The ESI–MS signal intensity ratios of the four phosphopeptide standards labeled with N-acetoxy-H3-succinimide (H3-NAS) and N-acetoxy-D3-succinimide (D3-NAS), following TiO2/MHMSM capture are linearly correlated with the molar ratios of the “light” to “heavy” phosphopeptides over the range of 0.1–4 with an r 2 of up to 0.998 and a slope of close to 1. The recovery of the four phosphopeptides spiked at low, medium and high levels in human sera were 98.4–111.9% with RSDs ranging 2.0–10.1%. The absolute quantification of the phosphopeptides in serum samples of 20 healthy persons and 20 gastric cancer patients by the developed method demonstrated that 3 out of the 4 phosphopeptides showed remarkable variation in serum level between healthy and cancer groups, and the phosphopeptide DpSGEGDFLAEGGGVR is significantly down-regulated in the serum of patients, being a potential biomarker for gastric cancer diagnosis. [Copyright &y& Elsevier]

The organometallic ruthenium(II) anticancer complexes [(η6-arene)Ru(en)Cl]+ (arene= p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en=ethylenediamine), exhibit in vitro and in vivo anticancer activities. In the present work, we show that they inhibit human glutathione-S-transferase π (GSTπ) with IC50 values of 59.4±1.3, 63.2±0.4 and 37.2±1.1μM, respectively. Mass spectrometry revealed that complex 1 binds to the S-donors of Cys15, Cys48 within the G-site and Cys102 at the interface of the GSTπ dimer, while complex 2 binds to Cys48 and Met92 at the dimer interface and complex 3 to Cys15, Cys48 and Met92. Moreover, the binding of complex 1 to Cys15 and Cys102, complex 2 to Cys48 and complex 3 to Cys15 induces the irreversible oxidation of the coordinated thiolates to sulfenates. Molecular modeling studies indicate that the coordination of the {(arene)Ru(en)}2+ fragment to Cys48 blocks the hydrophilic G-site sterically, perhaps preventing substrate from proper positioning and accounting for the reduction in enzymatic activity of ruthenated GSTπ. The binding of the ruthenium arene complexes to Cys102 or Met92 disrupts the dimer interface which is an essential structural feature for the proper functioning of GSTπ, perhaps also contributing to the inhibition of GSTπ. [ABSTRACT FROM AUTHOR]

Abstract: The cyclic oxidation and reduction of methionine (Met) containing peptides and proteins play important roles in biological system. This work was contributed to analysis the cyclic oxidation and reduction processes of a methionine containing peptide which is very likely to relate in the cell signal transduction pathways. To mimic the biological oxidation condition, hydrogen peroxide was used as the reactive oxygen species to oxidize the peptide. Reversed-phase high-performance liquid chromatography and mass spectrometry were employed to monitor the reactions and characterize the structural changes of the products. A rapid reduction procedure was developed by simply using KI as the reductant, which is green and highly efficient. By investigation of the cyclic oxidation and reduction process, our work provides a new perspective to study the function and mechanism of Met containing peptides and proteins during cell signaling processes as well as diseases. [Copyright &y& Elsevier]

Abstract: Metallothioneins (MTs) are a group of low molecular weight (6–7KDa) proteins featured by high cysteine content which allows the proteins to bind to a diverse range of metals. MTs, of which the gene transcription can be induced by a variety of stimuli including hormones, cytokines and metal ions, have important biological functions such as storage, trafficking and homeostasis of metal ions, detoxification of heavy metals, resistance to ionizing radiation, scavenging hydroxyl radical, etc. Importantly, it has been reported that MTs play a role in oncogenesis and cancer prognosis, and are implicated in resistance of cancer cells to anticancer metallodrug cisplatin. In this work, we present a comparative study on interactions between MTs and cisplatin and ruthenium arene anticancer complexes using MALDI-TOF-MS. The results show that cisplatin coordinates to MT-I and MT-II at either pH 3.0 or 7.4, and exhibits a higher affinity to MT-II than to MT-I. The MT–Pt complexes increase significantly in content with decrease in pH values of solutions, indicative that cisplatin competes with zinc for coordination to cysteine residues on MTs and interferes with the binding of zinc to the proteins. While the ruthenium arene anticancer complexes [(η6-arene)Ru(en)Cl]PF6 (arene=benzene or biphenyl, en=ethylenediamine) hardly bind to MTs at acidic pH and coordinate to MTs at a much lower level than cisplatin at neutral pH, which may account for the less toxicity and lack of cross-resistance to cisplatin for this class of ruthenium anticancer complexes. With the MT–Pt complexes as model protein complexes, DDT, the widely used unfolding thiol-containing agent in proteomic research was shown to reduce the coordination of platinum to MTs significantly, implying that DDT used as unfolding reagent at high concentration may cause dissociation of bound metallodrug and should be applied with care. [Copyright &y& Elsevier]