Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera.

Currently, there are no reliable biomarkers for the diagnosis of pancreatic cancer (PaC). Glycoproteomic approaches that analyze the glycan determinants on specific glycoproteins have proven useful to develop more specific cancer biomarkers than the corresponding protein levels. In PaC, mesothelin (MSLN) is a neo-expressed glycoprotein. MSLN glycosylation has not been described and could be altered in PaC. In this work, we aimed to characterize MSLN glycans from PaC cells and serum samples to assess their potential usefulness as PaC biomarkers. First, we analyzed MSLN glycans from PaC cell lines and then we developed an enzyme-linked lectin assay to measure core fucosylated-MSLN (Cf-MSLN) glycoforms. MSLN glycans from PaC cells were analyzed by glycan sequencing and through Western blotting with lectins. All of the cell lines secreted MSLN, with its three N-glycosylation sites occupied by complex-type N-glycans, which were mainly α2,3-sialylated, core fucosylated and highly branched. The Cf-MSLN glycoforms were quantified on PaC serum samples, and compared with MSLN protein levels. The Cf-MSLN was significantly decreased in PaC patients compared to control sera, while no differences were detected by using MSLN protein levels. In conclusion, Cf-MSLN glycoforms were differently expressed in PaC, which opens the way to further investigate their usefulness as PaC biomarkers.

Characterisation of the main PSA glycoforms in aggressive prostate cancer.

Serum levels of prostate specific antigen (PSA) are commonly used for prostate cancer (PCa) detection. However, their lack of specificity to distinguish benign prostate pathologies from PCa, or indolent from aggressive PCa have prompted the study of new non-invasive PCa biomarkers. Aberrant glycosylation is involved in neoplastic progression and specific changes in PSA glycosylation pattern, as the reduction in the percentage of α2,6-sialic acid (SA) are associated with PCa aggressiveness. In this study, we have characterised the main sialylated PSA glycoforms from blood serum of aggressive PCa patients and have compared with those of standard PSA from healthy individuals' seminal plasma. PSA was immunoprecipitated and α2,6-SA were separated from α2,3-SA glycoforms using SNA affinity chromatography. PSA N-glycans were released, labelled and analysed by hydrophilic interaction liquid chromatography combined with exoglycosidase digestions. The results showed that blood serum PSA sialylated glycoforms containing GalNAc residues were largely increased in aggressive PCa patients, whereas the disialylated core fucosylated biantennary structures with α2,6-SA, which are the major PSA glycoforms in standard PSA from healthy individuals, were markedly reduced in aggressive PCa. The identification of these main PSA glycoforms altered in aggressive PCa opens the way to design specific strategies to target them, which will be useful to improve PCa risk stratification.

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.

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.

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

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

Synthesis and Biological Evaluation of Ru(II) and Pt(II) Complexes Bearing Carboxyl Groups as Potential Anticancer Targeted Drugs.

The synthesis and characterization of Pt(II) (1 and 2) and Ru(II) arene (3 and 4) or polypyridine (5 and 6) complexes is described. With the aim of having a functional group to form bioconjugates, one uncoordinated carboxyl group has been introduced in all complexes. Some of the complexes were selected for their potential in photodynamic therapy (PDT). The molecular structures of complexes 2 and 5, as well as that of the sodium salt of the 4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine ligand (cptpy), were determined by X-ray diffraction. Different techniques were used to evaluate the binding capacity to model DNA molecules, and MTT cytotoxicity assays were performed against four cell lines. Compounds 3, 4, and 5 showed little tendency to bind to DNA and exhibited poor biological activity. Compound 2 behaves as bonded to DNA probably through a covalent interaction, although its cytotoxicity was very low. Compound 1 and possibly 6, both of which contain a cptpy ligand, were able to intercalate with DNA, but toxicity was not observed for 6. However, compound 1 was active in all cell lines tested. Clonogenic assays and apoptosis induction studies were also performed on the PC-3 line for 1. The photodynamic behavior for complexes 1, 5, and 6 indicated that their nuclease activity was enhanced after irradiation at λ = 447 nm. The cell viability was significantly reduced only in the case of 5. The different behavior in the absence or presence of light makes complex 5 a potential prodrug of interest in PDT. Molecular docking studies followed by molecular dynamics simulations for 1 and the counterpart without the carboxyl group confirmed the experimental data that pointed to an intercalation mechanism. The cytotoxicity of 1 and the potential of 5 in PDT make them good candidates for subsequent conjugation, through the carboxyl group, to "selected peptides" which could facilitate the selective vectorization of the complex toward receptors that are overexpressed in neoplastic cell lines.

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.

Comparative Study of Blood-Based Biomarkers, α2,3-Sialic Acid PSA and PHI, for High-Risk Prostate Cancer Detection.

Prostate Specific Antigen (PSA) is the most commonly used serum marker for prostate cancer (PCa), although it is not specific and sensitive enough to allow the differential diagnosis of the more aggressive tumors. For that, new diagnostic methods are being developed, such as PCA-3, PSA isoforms that have resulted in the 4K score or the Prostate Health Index (PHI), and PSA glycoforms. In the present study, we have compared the PHI with our recently developed PSA glycoform assay, based on the determination of the α2,3-sialic acid percentage of serum PSA (% α2,3-SA), in a cohort of 79 patients, which include 50 PCa of different grades and 29 benign prostate hyperplasia (BPH) patients. The % α2,3-SA could distinguish high-risk PCa patients from the rest of patients better than the PHI (area under the curve (AUC) of 0.971 vs. 0.840), although the PHI correlated better with the Gleason score than the % α2,3-SA. The combination of both markers increased the AUC up to 0.985 resulting in 100% sensitivity and 94.7% specificity to differentiate high-risk PCa from the other low and intermediate-risk PCa and BPH patients. These results suggest that both serum markers complement each other and offer an improved diagnostic tool to identify high-risk PCa, which is an important requirement for guiding treatment decisions.

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.

Synthetic lethal interaction of cetuximab with MEK1/2 inhibition in NRAS-mutant metastatic colorectal cancer.

KRAS mutations are an established predictor of lack of response to EGFR-targeted therapies in patients with metastatic colorectal cancer (mCRC). However, little is known about the role of the rarer NRAS mutations as a mechanism of primary resistance to the anti-EGFR monoclonal antibody cetuximab in wild-type KRAS mCRC. Using isogenic mCRC cells with a heterozygous knock-in of the NRAS activating mutation Q61K, we aimed to elucidate the mechanism(s) by which mutant NRAS blocks cetuximab from inhibiting mCRC growth. NRASQ61K/+ cells were refractory to cetuximab-induced growth inhibition. Pathway-oriented proteome profiling revealed that cetuximab-unresponsive ERK1/2 phosphorylation was the sole biomarker distinguishing cetuximab-refractory NRASQ61K/+ from cetuximab-sensitive NRAS+/+ cells. We therefore employed four representative MEK1/2 inhibitors (binimetinib, trametinib, selumetinib, and pimasertib) to evaluate the therapeutic value of MEK/ERK signaling in cetuximab-refractory NRAS mutation-induced mCRC. Co-treatment with an ineffective dose of cetuximab augmented, up to more than 1,300-fold, the cytotoxic effects of pimasertib against NRASQ61K/+ cells. Simultaneous combination of MEK1/2 inhibitors with cetuximab resulted in extremely high and dose-dependent synthetic lethal effects, which were executed, at least in part, by exacerbated apoptotic cell death. Dynamic monitoring of real-time cell growth rates confirmed that cetuximab synergistically sensitized NRASQ61K/+ cellsto MEK1/2 inhibition. Our discovery of a synthetic lethal interaction of cetuximab in combination with MEK1/2 inhibition for the NRAS mutant subgroup of mCRC underscores the importance of therapeutic intervention both in the MEK-ERK and EGFR pathways to achieve maximal therapeutic efficacy against NRAS-mutant mCRC tumors.

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.

Improved Pancreatic Adenocarcinoma Diagnosis in Jaundiced and Non-Jaundiced Pancreatic Adenocarcinoma Patients through the Combination of Routine Clinical Markers Associated to Pancreatic Adenocarcinoma Pathophysiology.

BACKGROUND: There is still no reliable biomarker for the diagnosis of pancreatic adenocarcinoma. Carbohydrate antigen 19-9 (CA 19-9) is a tumor marker only recommended for pancreatic adenocarcinoma follow-up. One of the clinical problems lies in distinguishing between this cancer and other benign pancreatic diseases such as chronic pancreatitis. In this study we will assess the value of panels of serum molecules related to pancreatic cancer physiopathology to determine whether alone or in combination could help to discriminate between these two pathologies. METHODS: CA 19-9, carcinoembryonic antigen (CEA), C-reactive protein, albumin, insulin growth factor-1 (IGF-1) and IGF binding protein-3 were measured using routine clinical analyzers in a cohort of 47 pancreatic adenocarcinoma, 20 chronic pancreatitis and 15 healthy controls. RESULTS: The combination of CA 19-9, IGF-1 and albumin resulted in a combined area under the curve (AUC) of 0.959 with 93.6% sensitivity and 95% specificity, much higher than CA 19-9 alone. An algorithm was defined to classify the patients as chronic pancreatitis or pancreatic cancer with the above specificity and sensitivity. In an independent validation group of 20 pancreatic adenocarcinoma and 13 chronic pancreatitis patients, the combination of the four molecules classified correctly all pancreatic adenocarcinoma and 12 out of 13 chronic pancreatitis patients. CONCLUSIONS: Although this panel of markers should be validated in larger cohorts, the high sensitivity and specificity values and the convenience to measure these parameters in clinical laboratories shows great promise for improving pancreatic adenocarcinoma diagnosis.

A comparison of non-biologically active truncated EGF (EGFt) and full-length hEGF for delivery of Auger electron-emitting 111In to EGFR-positive breast cancer cells and tumor xenografts in athymic mice.

INTRODUCTION: EGFt is a truncated form of human epidermal growth factor (hEGF) that is non-biologically active but retains binding and internalization into EGFR-positive cells. Our aim was to compare EGFt and hEGF for delivery of (111)In to human breast cancer (BC) cells and tumors and evaluate its cytotoxicity against EGFR-positive BC cells, mediated by the Auger electron emissions of (111)In. METHODS: The binding, internalization and nuclear localization of EGFt and hEGF in MDA-MB-468 human BC cells were first assessed by confocal fluorescence microscopy. Subcellular fractionation was then used to quantify the cellular and nuclear uptake of (111)In-EGFt and (111)In-hEGF in MDA-MB-468 cells. The effect of exposure in vitro to (111)In-EGFt or (111)In-hEGF on the clonogenic survival of MDA-MB-468 (10(6) EGFR/cell) or MCF-7 cells (10(4) EGFR/cell) was determined. The pharmacokinetics and tumor and normal tissue biodistribution of (111)In-EGFt was compared to (111)In-hEGF in CD-1 athymic mice with s.c. MDA-MB-468 and MCF-7 tumors. Nuclear importation in MDA-MB-468 tumors was determined ex vivo by subcellular fractionation. RESULTS: Fluorescently-labeled EGFt and hEGF were bound, internalized and localized in the nucleus of MDA-MB-468 cells. Binding of (111)In-EGFt to MDA-MB-468 cells was 8-fold lower than (111)In-hEGF, but nuclear importation as a proportion of cell-bound (111)In was 3.6-fold greater than (111)In-hEGF. Nuclear uptake of (111)In-EGFt was lower than (111)In-hEGF when differences in cell binding were taken into account. The cytotoxicity of (111)In-EGFt (1.0MBq/mL; 10 nmols/L) against MDA-MB-468 cells was 9-fold lower than (111)In-hEGF but only 2-fold lower at a higher concentration (1.85 MBq/mL; 40 nmols/L). (111)In-EGFt and (111)In-hEGF exhibited greater cytotoxicity against MDA-MB-468 cells than MCF-7 cells. (111)In-EGFt was eliminated more slowly from the blood of tumor-bearing mice and exhibited lower liver uptake but higher kidney accumulation. Uptake of (111)In-EGFt in MDA-MB-468 tumors was 2.2-fold lower than (111)In-hEGF, and was blocked by anti-EGFR monoclonal antibody, nimotuzumab. Nuclear uptake into MDA-MB-468 tumor cells was higher for (111)In-EGFt than (111)In-hEGF, but when the lower tumor uptake of (111)In-EGFt was considered, there were no overall differences. CONCLUSION: We conclude that the absence of biological activity of EGFt makes it attractive for delivery of Auger electron-emitting (111)In to EGFR-overexpressing BC, but its lower cellular and tumor uptake would limit its effectiveness compared to (111)In-hEGF. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: (111)In-EGFt may reduce the adverse effects previously observed in patients administered (111)In-hEGF since it is not biologically active, but its lower uptake by BC cells and tumors would limit its effectiveness for treatment of breast cancer.

Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.

Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.

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.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Identification of caveolin-1 as a potential causative factor in the generation of trastuzumab resistance in breast cancer cells.

The oncogenic tyrosine kinase receptor ErbB2 is a prognostic factor and target for breast cancer therapeutics. In contrast with the other ErbB receptors, ErbB2 is hardly internalized by ligand induced mechanisms, indicating a prevalent surface expression. Elevated levels of ErbB2 in tumor cells are associated with its defective endocytosis and down regulation. Here we show that caveolin-1 expression in breast cancer derived SKBR-3 cells (SKBR-3/Cav-1) facilitates ligand induced ErbB2 endocytosis using an artificial peptide ligand EC-eGFP. Similarly, stimulation with humanized anti ErbB2 antibody Trastuzumab (Herceptin) was found to be internalized and co-localized with caveolin-1 in SKBR-3/Cav-1 cells. Internalized EC-eGFP and Trastuzumab in SKBR-3/Cav-1 cells were then delivered via caveolae to the caveolin-1 containing early endosomes. Consequently, attenuated Fc receptor mediated ADCC functions were observed when exposed to Trastuzumab and EC-Fc (EC-1 peptide conjugated to Fc part of human IgG). On the other hand, this caveolae dependent endocytic synergy was not observed in parental SKBR-3 cells. Therefore, caveolin-1 expression in breast cancer cells could be a predictive factor to estimate how cancer cells are likely to respond to Trastuzumab treatment.

α2,3-Sialyltransferase ST3Gal IV promotes migration and metastasis in pancreatic adenocarcinoma cells and tends to be highly expressed in pancreatic adenocarcinoma tissues.

Sialyltransferases have received much attention recently as they are frequently up-regulated in cancer cells. However, the role played by each sialyltransferase in tumour progression is still unknown. α2,3-Sialyltransferases ST3Gal III and ST3Gal IV are involved in sialyl-Lewis(x) (SLe(x)) synthesis. Given that the role of ST3Gal III in pancreatic adenocarcinoma cells has been previously reported, in this study we have focused on investigating the role of ST3Gal IV in the acquisition of adhesive, migratory and metastatic capabilities and, secondly, in analyzing the expression of ST3Gal III and ST3Gal IV in pancreatic adenocarcinoma tissues versus control tissues. ST3Gal IV overexpressing pancreatic adenocarcinoma MDAPanc-28 cell lines were generated. They showed a heterogeneous increase in SLe(x), and enhanced E-selectin adhesion and migration. Furthermore, when injected into nude mice, increased metastasis and decreased survival were found in comparison with controls. The behaviour of MDAPanc-28 ST3Gal IV overexpressing cells in these processes was similar to the already reported MDAPanc-28 ST3Gal III overexpressing cells. Furthermore, pancreatic adenocarcinoma tissues tended to express high levels of ST3Gal III and ST3Gal IV together with other fucosyltransferase genes FUT3 and FUT6, all involved in the last steps of sialyl-Lewis(x) biosynthesis. In conclusion, both α2,3-sialyltransferases are involved in key steps of pancreatic tumour progression processes and are highly expressed in most pancreatic adenocarcinoma tissues.

Preparation of new half sandwich ruthenium arene complexes with aminophosphines as potential chemotherapeutics.

Aminophosphines 2-(diphenylphosphino)-1-methylimidazole (dpim) and diphenyl-2-pyridylphosphine (PPh(2)py) have been used to prepare two series of Ru(II) arene complexes of formulae [(η(6)-p-cymene)Ru(κ(2)-O,O'-X)(κ(1)-P-dpim)]Y (series a: 1a·Y-3a·Y) and [(η(6)-p-cymene)Ru(κ(2)-O,O'-X)(κ(1)-P-PPh(2)py)]Y (series b: 1b·Y-3b·Y) (where X=acac, acetylacetonate; bzac, benzoyl acetonate; dbzm, dibenzoyl methanoate; Y=BF(4), BPh(4)). The structures of 1a·BF(4), 1a·BPh(4), 3a·BF(4), 1b·BPh(4) and 3b·BPh(4) were determined by X-ray diffraction. The tetrafluoroborate derivatives are more soluble in organic solvents than their tetraphenylborate counterparts. Five BF(4)(-) derivatives (all except the unstable 1b·BF(4)) were selected to evaluate the cytotoxic behavior in vitro against the human cancer cell lines MCF-7 (breast cancer) and CAPAN-1 (pancreatic cancer). 2b·BF(4) and 3b·BF(4) exhibited IC(50) values similar to those of cisplatin. Electrophoresis and AFM studies showed good correspondence between the biological activity levels of 2b·BF(4) and 3b·BF(4) and their ability to modify the DNA structure. Hydrolytic studies indicate that aquation could be involved in the activation mechanism of these complexes and confirm that the hydrolysis rate of 3b·BF(4) is higher than that of 3a·BF(4). Thus, the cytotoxic activity trends are explained in terms of the higher reactivity of derivatives from series b, which in turn is rationalized as being the result of the electronic features of dpim and PPh(2)py established by cyclic voltammetry measurements.

Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes.

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl(2)(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η(6)-bip)Os(4-CO(2)-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η(6)-p-cym)RuCl(dap)](+) (p-cym = p-cymene) (5), and [(η(6)-p-cym)RuCl(imidazole-CO(2)H)(PPh(3))](+) (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC(50) = 63 ± 2 µM in MCF-7 cells and IC(50) = 26 ± 3 µM in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC(50) = 45 ± 2.6 µM in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically.