Ir(III) Half-Sandwich Photosensitizers with a π-Expansive Ligand for Efficient Anticancer Photodynamic Therapy.

One approach to reduce the side effects of chemotherapy in cancer treatment is photodynamic therapy (PDT), which allows spatiotemporal control of the cytotoxicity. We have used the strategy of coordinating π-expansive ligands to increase the excited state lifetimes of Ir(III) half-sandwich complexes in order to facilitate the generation of 1O2. We have obtained derivatives of formulas [Cp*Ir(C∧N)Cl] and [Cp*Ir(C∧N)L]BF4 with different degrees of π-expansion in the C∧N ligands. Complexes with the more π-expansive ligand are very effective photosensitizers with phototoxic indexes PI > 2000. Furthermore, PI values of 63 were achieved with red light. Time-dependent density functional theory (TD-DFT) calculations nicely explain the effect of the π-expansion. The complexes produce reactive oxygen species (ROS) at the cellular level, causing mitochondrial membrane depolarization, cleavage of DNA, nicotinamide adenine dinucleotide (NADH) oxidation, as well as lysosomal damage. Consequently, cell death by apoptosis and secondary necrosis is activated. Thus, we describe the first class of half-sandwich iridium cyclometalated complexes active in PDT.

Rational design of mitochondria targeted thiabendazole-based Ir(III) biscyclometalated complexes for a multimodal photodynamic therapy of cancer.

Despite their outstanding properties as potential photosensitizers for photodynamic therapy (PDT), Ir(III) biscyclometalated complexes need both further developments to overcome remaining limitations and in-depth investigations into their mechanisms of action to reach clinic application in the treatment of cancer. This work describes the synthesis of a family of Ir(III) complexes of general formula [Ir(C^N)2(N^N')]Cl (N^N' = thiabendazole-based ligands; C^N = ppy (2-phenylpyridinate) (Series A), or dfppy (2-(2,4-difluorophenyl)pyridinate) (Series B)) and their evaluation as potential PDT agents. These complexes are partially soluble in water and exhibit cytotoxic activity in the absence of light irradiation versus several cancer cell lines. Furthermore, the cytotoxic activity of derivatives of Series A is enhanced upon irradiation, particularly for complexes [1a]Cl and [3a]Cl, which show phototoxicity indexes (PI) above 20. Endocytosis was established as the uptake mechanism for [1a]Cl and [3a]Cl in prostate cancer cells by flow cytometry. These derivatives mainly accumulate in the mitochondria as shown by colocalization confocal microscopy experiments. Presumably, [1a]Cl and [3a]Cl induce death on cancer cells under irradiation through apoptosis triggered by a multimodal mechanism of action, which likely involves damage over mitochondrial DNA and mitochondrial membrane depolarization. Both processes seem to be the result of photocatalytic oxidation processes.

Photodynamic therapy with mitochondria-targeted biscyclometallated Ir(III) complexes. Multi-action mechanism and strong influence of the cyclometallating ligand.

Photodynamic therapy is an alternative to classical chemotherapy due to its potential to reduce side effects by a controlled activation of a photosensitizer through local irradiation with light. The photosensitizer then interacts with oxygen and generates reactive oxygen species. Iridium biscyclometallated complexes are very promising photosensitizers due to their exceptional photophysical properties and their ability to target mitochondria. Four Ir(III) biscyclometallated complexes of formula [Ir(C^N)2(N^N')]Cl, where N^N' is a ligand containing a benzimidazolyl fragment, have been synthesized and characterized. The C^N ligands were 2-phenylpyridinate (ppy) and 2-(2,4-difluorophenyl)pyridinate (dfppy). The complexes exhibited high photostability. The electrochemical and photophysical properties were modulated by both the cyclometallating and the ancillary ligands. The dfppy derivatives yielded the highest emission energy values, quantum yields of phosphorescence and excited state lifetimes. All complexes generated 1O2 in aerated solutions upon irradiation. Biological studies revealed that these complexes have a moderate cytotoxicity in the dark against different human cancer cell lines: prostate (PC-3), colon (CACO-2) and melanoma (SK-MEL-28), and against non-malignant fibroblasts (CCD-18Co). However, derivatives with ppy ligands ([1a]Cl, [2a]Cl) yielded a relevant photodynamic activity upon light irradiation (450 nm, 24.1 J cm-2), with phototoxicity indexes (EC50,dark/EC50,light) of 20.8 and 17.3, respectively, achieved in PC-3 cells. Mechanistic studies showed that these complexes are taken up by the cells through endocytosis and preferentially accumulate in mitochondria. Upon photoactivation, the complexes induced mitochondrial membrane depolarization and DNA damage, thus triggering cell death, mainly by apoptosis. Complex [1a]Cl is also able to oxidize NADH. This mitochondria-targeted photodynamic mechanism greatly inhibited the reproductive capacity of cancer cells and provides a valuable alternative to traditional chemotherapy for the controlled treatment of cancer.

Effect of the aniline fragment in Pt(II) and Pt(IV) complexes as anti-proliferative agents. Standard reduction potential as a more reliable parameter for Pt(IV) compounds than peak reduction potential.

The problems of resistance and side effects associated with cisplatin and other chemotherapeutic drugs have boosted research aimed at finding new compounds with improved properties. The use of platinum(IV) prodrugs is one alternative, although there is some controversy regarding the predictive ability of the peak reduction potentials. In the work described here a series of fourteen chloride Pt(II) and Pt(IV) compounds was synthesised and fully characterised. The compounds contain different bidentate arylazole heterocyclic ligands. Their cytotoxic properties against human lung carcinoma (A549), human breast carcinoma (MCF7) and human colon carcinoma (HCT116 and HT29) cell lines were studied. A clear relationship between the type of ligand and the anti-proliferative properties was found, with the best results obtained for the Pt(II) compound that contains an aniline fragment, (13), thus evidencing a positive effect of the NH2 group. Stability and aquation studies in DMSO, DMF and DMSO/water mixtures were carried out on the active complexes and an in-depth analysis of the two aquation processes, including DFT analysis, of 13 was undertaken. It was verified that DNA was the target and that cell death occurred by apoptosis in the case of 13. Furthermore, the cytotoxic derivatives did not exhibit haemolytic activity. The reduction of the Pt(IV) compounds whose Pt(II) congeners were active was studied by several techniques. It was concluded that the peak reduction potential was not useful to predict the ability for reduction. However, a correlation between the cytotoxic activity and the standard reduction potential was found.

A nucleus-directed bombesin derivative for targeted delivery of metallodrugs to cancer cells.

We have synthesized a set of bombesin derivatives with the aim of exploring their tumor targeting properties to deliver metal-based chemotherapeutics into cancer cells. Peptide QRLGNQWAVGHLL-NH2 (BN3) was selected based on its high internalization in gastrin-releasing peptide receptor (GRPR)-overexpressing PC-3 cells. Three metallopeptides were prepared by incorporating the terpyridine Pt(II) complex [PtCl(cptpy)]Cl (1) (cptpy = 4'-(4-carboxyphenyl)-2,2':6,2″-terpyridine) at the N-terminus of BN3 or at the NƐ- or Nα-amino group of an additional Lys residue (1-BN3, Lys-1-BN3 and 1-Lys-BN3, respectively). 1-Lys-BN3 displayed the best cytotoxic activity (IC50: 19.2 ±â€¯1.7 µM) and similar ability to intercalate into DNA than complex 1. Moreover, the polypyridine Ru(II) complex [Ru(bpy)2)(cmbpy)](PF6)2 (2) (bpy = 2,2'-bipyridine; cmbpy = 4-methyl-2,2'-bipyridine-4'-carboxylic acid), with proven activity as photosensitizer, was coupled to BN3 leading to metallopeptide 2-Lys-BN3. Upon photoactivation, 2-Lys-BN3 displayed 2.5-fold higher cytotoxicity against PC-3 cells (IC50: 7.6 ±â€¯1.0 µM) than complex 2. To enhance the accumulation of the drugs into the cell nucleus, the nuclear localization signal (NLS) PKKKRKV was incorporated at the N-terminus of BN3. NLS-BN3 displayed higher cellular internalization along with nuclear biodistribution. Accordingly, metallopeptides 1-NLS-BN3 and 2-NLS-BN3 showed increased cytotoxicity (IC50: 12.0 ±â€¯1.1 µM and 2.3 ±â€¯1.1 µM). Interestingly, the phototoxic index of 2-NLS-BN3 was 8-fold higher than that of complex 2. Next, the selectivity towards cancer cells was explored using 1BR3.G fibroblasts. Higher selectivity indexes were obtained for 1-NLS-BN3 and 2-NLS-BN3 than for the unconjugated complexes. These results prove NLS-BN3 effective for targeted delivery of metallodrugs to GRPR-overexpressing cells and for enhancing the cytotoxic efficacy of metal-based photosensitizers.

Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p < 0.028), whereas in AIEC strains ompA expression was the only increased when growing in association with cells (p = 0.032), but they did not significantly alter ompC and ompF expression under this condition (p > 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains.

Multivariate data analysis for the detection of human alpha-acid glycoprotein aberrant glycosylation in pancreatic ductal adenocarcinoma.

Relative quantification of human alpha-acid glycoprotein (hAGP) glycan isomers using [12C6]/[13C6]-aniline in combination with multivariate data analysis is proposed as an efficient method for the identification of pancreatic ductal adenocarcinoma (PDAC) glycan biomarkers in serum samples. Intact and desialylated glycans from hAGP, purified from serum samples of patients with PDAC and chronic pancreatitis (ChrP), were labeled with aniline and analyzed by µZIC-HILIC-MS. Afterwards, partial least squares discriminant analysis (PLS-DA) was applied to the relative areas obtained for all glycan isomers in the different samples: pathological (ChrP or PDAC) versus healthy samples. Seven intact glycan isomers with α2-6 linked sialic acids, five of them also fucosylated, were the most meaningful to distinguish between PDAC and ChrP patients. The desialylated glycan isomers also identified by PLS-DA as potential biomarker candidates confirmed that antenna but also core fucosylation could be involved in PDAC. The analysis of intact and desialylated glycan isomers in combination with the multivariate data analysis revealed that the triantennary glycan with two fucoses of hAGP could have in the future a relevant role in the differentiation of patients with PDAC from those with ChrP. SIGNIFICANCE: Multivariate data analysis is currently being used in many omics fields for biomarker discovery. However, to date, no glycomics studies have applied chemometric tools combined with mass spectrometry in a preclinical research. In this work, this methodology has been used to identify altered glycosylation of human alpha-acid glycoprotein in pancreatic ductal adenocarcinoma (PDAC). The obtained results reveal that the triantennary glycan with two fucoses could have a great biomarker potential as it was relevant to differentiate PDAC and chronic pancreatitis (ChrP) patients.

Glycoprotein biomarkers for the detection of pancreatic ductal adenocarcinoma.

Pancreatic cancer (PaC) shows a clear tendency to increase in the next years and therefore represents an important health and social challenge. Currently, there is an important need to find biomarkers for PaC early detection because the existing ones are not useful for that purpose. Recent studies have indicated that there is a large window of time for PaC early detection, which opens the possibility to find early biomarkers that could greatly improve the dismal prognosis of this tumor. The present manuscript reviews the state of the art of the existing PaC biomarkers. It focuses on the anomalous glycosylation process and its role in PaC. Glycan structures of glycoconjugates such as glycoproteins are modified in tumors and these modifications can be detected in biological fluids of the cancer patients. Several studies have found serum glycoproteins with altered glycan chains in PaC patients, but they have not shown enough specificity for PaC. To find more specific cancer glycoproteins we propose to analyze the glycan moieties of a battery of glycoproteins that have been reported to increase in PaC tissues and that can also be found in serum. The combination of these new candidate glycoproteins with their aberrant glycosylation together with the existing biomarkers could result in a panel, which would expect to give better results as a new tool for early diagnosis of PaC and to monitor the disease.

Analysis of sialyl-Lewis x on MUC5AC and MUC1 mucins in pancreatic cancer tissues.

Pancreatic adenocarcinoma (PDAC) lacks efficient biomarkers. Mucins are glycoproteins that can carry aberrant glycosylation in cancer. Our objective was to identify cancer-related glycan epitopes on MUC1 and MUC5AC mucins in PDAC as potential biomarkers. We have analysed the tumour-associated carbohydrate antigens sialyl-Lewis x (SLex) and sialyl-Tn (STn) on MUC1 and MUC5AC in PDAC tissues. The selected cohort for this study consisted of twenty-one PDAC tissues positive for SLex antigen and three normal pancreas specimens as controls. STn expression was shown in 76% of the PDAC tissues. MUC1 and MUC5AC were detected in 90% of PDAC tissues. We performed in situ proximity ligation assay combining antibodies against mucins and glycan epitopes to identify specific mucin glycoforms. MUC1-SLex and MUC5AC-SLex were found in 68% and 84% respectively, of the mucin expressing PDAC tissues, while STn hardly colocalized with any of the evaluated mucins. Further analysis by Western blot of MUC5AC and SLex in eight PDAC tissue lysates showed that six out of eight cases were positive for both markers. Moreover, immunoprecipitation of MUC5AC from positive PDAC tissues and subsequent SLex immunodetection confirmed the presence of SLex on MUC5AC. Altogether, MUC5AC-SLex glycoform is present in PDAC and can be regarded as potential biomarker.

Erratum: Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes: Erratum.

[This corrects the article DOI: 10.7150/thno.15226.].

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer.

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA.

Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins.

Comparative analysis of prostate-specific antigen by two-dimensional gel electrophoresis and capillary electrophoresis.

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes.

New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the α2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the α2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic.

Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in pancreatic cancer.

Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (µZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker.

Identification of potential pancreatic cancer serum markers: Increased sialyl-Lewis X on ceruloplasmin.

Pancreatic adenocarcinoma (PDAC) usually shows an enhanced expression of sialyl-Lewis X (sLe(x)) and related epitopes. PDAC may secrete some of the proteins carrying such increased sLe(x) determinant into serum, so they could be used as PDAC markers. Previously, we identified acute-phase proteins with increased sLe(x) in both PDAC and in chronic pancreatitis patients. In this study, depleted sera from the main acute-phase proteins has been analysed for the search of proteins with increased sLe(x) levels in PDAC. Sera from healthy controls, chronic pancreatitis and PDAC patients were depleted, electrophoresed and subjected to sLe(x) immunodetection. Proteins that differentially expressed sLe(x) in PDAC were trypsin digested and identified by LC-ESI-QTOF mass spectrometry. Five protein bands that differentially expressed sLe(x) in PDAC were identified and corresponded to seven different acute-phase proteins. Among them, ceruloplasmin (CP) was selected for further analysis. N-glycan sequencing of CP confirmed the increase of sLe(x) levels in CP in PDAC patients. Healthy controls, chronic pancreatitis and PDAC patients' sera were immunoprecipitated with anti-CP antibodies, and their sLe(x) and CP levels were analysed by western blot. The sLe(x)/CP ratio tended to be higher for the PDAC group, which altogether suggests that the sLe(x)/CP ratio could be a useful biomarker for PDAC.

Integrin-targeted delivery into cancer cells of a Pt(IV) pro-drug through conjugation to RGD-containing peptides.

Conjugates of a Pt(IV) derivative of picoplatin with monomeric (Pt-c(RGDfK), 5) and tetrameric (Pt-RAFT-{c(RGDfK)}4, 6) RGD-containing peptides were synthesized with the aim of exploiting their selectivity and high affinity for αVß3 and αVß5 integrins for targeted delivery of this anticancer metallodrug to tumor cells overexpressing these receptors. Solid- and solution-phase approaches in combination with click chemistry were used for the preparation of the conjugates, which were characterized by high resolution ESI MS and NMR. αVß3 and αVß5 integrin expression was evaluated in a broad panel of human cancer and non-malignant cells. SK-MEL-28 melanoma cells were selected based on the high expression levels of both integrins, while CAPAN-1 pancreatic cancer cells and 1BR3G fibroblasts were selected as the negative control. Internalization experiments revealed a good correlation between integrin expression and the cellular uptake of the corresponding fluorescein-labeled peptides and that the internalization capacity of the tetrameric RGD-containing peptide was considerably higher than that of the monomeric one. Cytotoxic experiments indicated that the antitumor activity of picoplatin in melanoma cells was increased by 2.6-fold when its Pt(IV) derivative was conjugated to c(RGDfK) (IC50 = 12.8 ± 2.1 µM) and by 20-fold when conjugated to RAFT-{c(RGDfK)}4 (IC50 = 1.7 ± 0.6 µM). In contrast, the cytotoxicity of the conjugates was inhibited in control cells lacking αVß3 and αVß5 integrin expression. Finally, cellular uptake studies by ICP-MS confirmed a good correlation between the levels of expression of integrins, intracellular platinum accumulation and antitumor activity. Indeed, accumulation and cytotoxicity were much higher in SK-MEL-28 cells than in CAPAN-1, being particularly higher in the case of the tetrameric conjugate. The overall results highlight that the great ability of RAFT-{c(RGDfK)}4 to bind to and to be internalized by integrins overexpressed in SK-MEL-28 cells results in higher accumulation of the Pt(IV) complex, leading to a high antitumor activity. These studies provide new insights into the potential of targeting αVß3 and αVß5 integrins with Pt(IV) anticancer pro-drugs conjugated to tumor-targeting devices based on RGD-containing peptides, particularly on how multivalency can improve both the selectivity and potency of such metallodrugs by increasing cellular accumulation in tumor tissues.

Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresis.

2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, α1-antitrypsin and ribonuclease 1), which N-glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule.

Altered glycosylation in tumours focused to cancer diagnosis.

The lack of specific and sensitive tumour markers for early detection of cancer is driving a search for new approaches that could identify biomarkers. Markers are needed to alert clinicians at the early stages of tumourogenesis, before the cancer has metastasized, when the therapeutic drugs are more effective. Most tumour markers currently used in clinics are serum glycoproteins, frequently highly glycosylated mucins. Typically, the disease marker is the protein and not the glycan moiety of the corresponding glycoprotein or mucin. The increasing knowledge of the role of glycans in cancer suggests that further studies may assist both in determining their role in every step of tumour progression, and in the design of new therapeutic and diagnosic approaches. Detection of the altered glycans in serum tumour glycoproteins could be a way to achieve specificity in tumour detection. In this review, we focus on the glycan changes of two serum glycoproteins, prostate specific antigen--currently used as a tumour marker of prostate cancer--and human pancreatic ribonuclease in pancreatic adenocarcinoma. The detection of glycan changes, associated with subsets of glycoforms in serum glycoproteins that are specific to the tumour situation, could be the basis for developing more specific biomarkers.

Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer.

Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.