Knockdown of α2,3-Sialyltransferases Impairs Pancreatic Cancer Cell Migration, Invasion and E-selectin-Dependent Adhesion.

Aberrant sialylation is frequently found in pancreatic ductal adenocarcinoma (PDA). α2,3-Sialyltransferases (α2,3-STs) ST3GAL3 and ST3GAL4 are overexpressed in PDA tissues and are responsible for increased biosynthesis of sialyl-Lewis (sLe) antigens, which play an important role in metastasis. This study addresses the effect of α2,3-STs knockdown on the migratory and invasive phenotype of PDA cells, and on E-selectin-dependent adhesion. Characterization of the cell sialome, the α2,3-STs and fucosyltransferases involved in the biosynthesis of sLe antigens, using a panel of human PDA cells showed differences in the levels of sialylated determinants and α2,3-STs expression, reflecting their phenotypic heterogeneity. Knockdown of ST3GAL3 and ST3GAL4 in BxPC-3 and Capan-1 cells, which expressed moderate to high levels of sLe antigens and α2,3-STs, led to a significant reduction in sLex and in most cases in sLea, with slight increases in the α2,6-sialic acid content. Moreover, ST3GAL3 and ST3GAL4 downregulation resulted in a significant decrease in cell migration and invasion. Binding and rolling to E-selectin, which represent key steps in metastasis, were also markedly impaired in the α2,3-STs knockdown cells. Our results indicate that inhibition of ST3GAL3 and ST3GAL4 may be a novel strategy to block PDA metastasis, which is one of the reasons for its dismal prognosis.

Solid-Phase Approaches for Labeling Targeting Peptides with Far-Red Emitting Coumarin Fluorophores.

Fluorophores based on organic molecules hold great potential for ligand-targeted imaging applications, particularly those operating in the optical window in biological tissues. In this work, we have developed three straightforward solid-phase approaches based on amide-bond formation or a Cu(I)-catalyzed azide-alkyne click (CuAAC) reaction for labeling an octreotide peptide with far-red emitting coumarin-based COUPY dyes. First, the conjugatable versions of COUPY fluorophores incorporating the required functional groups (e.g., carboxylic acid, azide, or alkyne) were synthesized and characterized. All of them were found fully compatible with Fmoc/ tBu solid-phase peptide synthesis, which allowed for the labeling of octreotide either through amide-bond formation or by CuAAC reaction. A near quantitative conversion was obtained after only 1 h of reaction at RT when using CuSO4 and sodium ascorbate independently of the click chemistry approach used (azido-COUPY/alkynyl-peptide resin or alkynyl-COUPY/azido-peptide resin). COUPY-octreotide conjugates were found stable in cell culture medium as well as noncytotoxic in HeLa cells, and their spectroscopic and photophysical properties were found similar to those of their parent coumarin dyes. Finally, the potential bioimaging applications of COUPY-octreotide conjugates were demonstrated by confocal microscopy through the visualization of living HeLa cells overexpressing the somatostatin subtype-2 receptor.

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.

A Green Light-Triggerable RGD Peptide for Photocontrolled Targeted Drug Delivery: Synthesis and Photolysis Studies.

We describe for the first time the synthesis and photochemical properties of a coumarin-caged cyclic RGD peptide and demonstrate that uncaging can be efficiently performed with biologically compatible green light. This was accomplished by using a new dicyanocoumarin derivative (DEAdcCE) for the protection of the carboxyl function at the side chain of the aspartic acid residue, which was selected on the basis of Fmoc-tBu SPPS compatibility and photolysis efficiency. The shielding effect of a methyl group incorporated in the coumarin derivative near the ester bond linking both moieties in combination with the use of acidic additives such as HOBt or Oxyma during the basic Fmoc-removal treatment were found to be very effective for minimizing aspartimide-related side reactions. In addition, a conjugate between the dicyanocoumarin-caged cyclic RGD peptide and ruthenocene, which was selected as a metallodrug model cargo, has been synthesized and characterized. The fact that green-light triggered photoactivation can be efficiently performed both with the caged peptide and with its ruthenocenoyl bioconjugate reveals great potential for DEAdcCE-caged peptide sequences as selective drug carriers in the context of photocontrolled targeted anticancer strategies.

A Photoactivatable Platinum(IV) Anticancer Complex Conjugated to the RNA Ligand Guanidinoneomycin.

A photoactivatable platinum(IV) complex, trans,trans,trans-[Pt(N3 )2 (OH)(succ)(py)2 ] (succ=succinylate, py=pyridine), has been conjugated to guanidinoneomycin to study the effect of this guanidinum-rich compound on the photoactivation, intracellular accumulation and phototoxicity of the pro-drug. Surprisingly, trifluoroacetic acid treatment causes the replacement of an azido ligand and the axial hydroxide ligand by trifluoroacetate, as shown by NMR spectroscopy, MS and X-ray crystallography. Photoactivation of the platinum-guanidinoneomycin conjugate in the presence of 5'-guanosine monophosphate (5'-GMP) led to the formation of trans-[Pt(N3 )(py)2 (5'-GMP)](+) , as does the parent platinum(IV) complex. Binding of the platinum(II) photoproduct {PtN3 (py)2 }(+) to guanine nucleobases in a short single-stranded oligonucleotide was also observed. Finally, cellular uptake studies showed that guanidinoneomycin conjugation improved the intracellular accumulation of the platinum(IV) pro-drug in two cancer cell lines, particularly in SK-MEL-28 cells. Notably, the higher phototoxicity of the conjugate in SK-MEL-28 cells than in DU-145 cells suggests a degree of selectivity towards the malignant melanoma cell line.

Design, Preparation, and Characterization of Zn and Cu Metallopeptides Based On Tetradentate Aminopyridine Ligands Showing Enhanced DNA Cleavage Activity.

The conjugation of redox-active complexes that can function as chemical nucleases to cationic tetrapeptides is pursued in this work in order to explore the expected synergistic effect between these two elements in DNA oxidative cleavage. Coordination complexes of biologically relevant first row metal ions, such as Zn(II) or Cu(II), containing the tetradentate ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane ((Me2)PyTACN) and (2S,2S')-1,1'-bis(pyrid-2-ylmethyl)-2,2'-bipyrrolidine ((S,S')-BPBP) have been linked to a cationic LKKL tetrapeptide sequence. Solid-phase synthesis of the peptide-tetradentate ligand conjugates has been developed, and the preparation and characterization of the corresponding metallotetrapeptides is described. The DNA cleavage activity of Cu and Zn metallopeptides has been evaluated and compared to their metal binding conjugates as well as to the parent complexes and ligands. Very interestingly, the oxidative Cu metallopeptides 1Cu and 2Cu show an enhanced activity compared to the parent complexes, [Cu(PyTACN)](2+) and [Cu(BPBP)](2+), respectively. Under optimized conditions, 1Cu displays an apparent pseudo first-order rate constant (kobs) of ∼0.16 min(-1) with a supercoiled DNA half-life time (t1/2) of ∼4.3 min. On the other hand, kobs for 2Cu has been found to be ∼0.11 min(-1) with t1/2 ≈ 6.4 min. Hence, these results point out that the DNA cleavage activities promoted by the metallopeptides 1Cu and 2Cu render ∼4-fold and ∼23 rate accelerations in comparison with their parent Cu complexes. Additional binding assays and mechanistic studies demonstrate that the enhanced cleavage activities are explained by the presence of the cationic LKKL tetrapeptide sequence, which induces an improved binding affinity to the DNA, thus bringing the metal ion, which is responsible for cleavage, in close proximity.

Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.

The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 µM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 µM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 µM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.

Identification of BP16 as a non-toxic cell-penetrating peptide with highly efficient drug delivery properties.

Antimicrobial peptides are an interesting source of non-cytotoxic drug delivery vectors. Herein, we report on the identification of a new cell-penetrating peptide (KKLFKKILKKL-NH2, BP16) from a set of antimicrobial peptides selected from a library of cecropin-melittin hybrids (CECMEL11) previously designed to be used in plant protection. This set of peptides was screened for their cytotoxicity against breast adenocarcinoma MCF-7, pancreas adenocarcinoma CAPAN-1 and mouse embryonic fibroblast 3T3 cell lines. BP16 resulted to be non-toxic against both malignant and non-malignant cells at concentrations up to 200 µM. We demonstrated by flow cytometry and confocal microscopy that BP16 is mainly internalized in the cells through a clathrin dependent endocytosis and that it efficiently accumulates in the cell cytoplasm. We confirmed that the cell-penetrating properties of BP16 are retained after conjugating it to the breast tumor homing peptide CREKA. Furthermore, we assessed the potential of BP16 as a drug delivery vector by conjugating the anticancer drug chlorambucil to BP16 and to a CREKA-BP16 conjugate. The efficacy of the drug increased between 6 and 9 times when conjugated to BP16 and between 2 and 4.5 times when attached to the CREKA-BP16 derivative. The low toxicity and the excellent cell-penetrating properties clearly suggest that BP16 is a suitable vector for the delivery of therapeutic agents into cells.

Towards efficient Ir(III) anticancer photodynamic therapy agents by extending π-conjugation on N

In this work we disclose a new family of biscyclometallated Ir(III) complexes of the general formula [Ir(C^N)2(N^N)]Cl (IrL1-IrL5), where HC^N is 1-phenyl-ß-carboline and N^N ligands (L1-L5) are different diimine ligands that differ from each other in the number of aromatic rings fused to the bipyridine scaffold. The photophysical properties of IrL1-IrL5 were thoroughly studied, and theoretical calculations were performed for a deeper comprehension of the respective variations along the series. All complexes exhibited high photostability under blue light irradiation. An increase in the number of aromatic rings led to a reduction in the HOMO-LUMO band gap causing a red-shift in the absorbance bands. Although all the complexes generated singlet oxygen (1O2) in aerated aqueous solutions through a photocatalytic process, IrL5 was by far the most efficient photosensitizer. Consequently, IrL5 was highly active in the photocatalytic oxidation of NADH. The formation of aggregates in DMSO at a high concentration (25 mM) was confirmed using different techniques, but was proved to be negligible in the concentration range of biological experiments. Moreover, ICP-MS studies proved that the cellular uptake of IrL2 and IrL3 is much better relative to that of IrL1, IrL4 and IrL5. The antiproliferative activity of IrL1-IrL5 was investigated in the dark and under blue light irradiation against different cancer cell lines. Complexes IrL1-IrL4 were found to be cytotoxic under dark conditions, while IrL5 turned out to be weakly cytotoxic. Despite the low cellular uptake of IrL5, this derivative exhibited a high increase of cytotoxicity upon blue light irradiation resulting in photocytotoxicity indexes (PI) up to 38. IrL1-IrL4 showed lower photocytotoxicity indexes ranging from 1.3 to 17.0. Haemolytic experiments corroborated the compatibility of our complexes with red blood cells. Confocal microscopy studies proved their accumulation in mitochondria, leading to mitochondrial membrane depolarization, and ruled out their localization in lysosomes. Overall, the mitochondria-targeted activity of IrL5, which inhibits considerably the viability of cancer cells upon blue light irradiation, allows us to outline this PS as a new alternative to traditional chemotherapeutic agents.

Insights into the anticancer photodynamic activity of Ir(III) and Ru(II) polypyridyl complexes bearing ß-carboline ligands.

Ir(III) and Ru(II) polypyridyl complexes are promising photosensitizers (PSs) for photodynamic therapy (PDT) due to their outstanding photophysical properties. Herein, one series of cyclometallated Ir(III) complexes and two series of Ru(II) polypyridyl derivatives bearing three different thiazolyl-ß-carboline N^N' ligands have been synthesized, aiming to evaluate the impact of the different metal fragments ([Ir(C^N)2]+ or [Ru(N^N)2]2+) and N^N' ligands on the photophysical and biological properties. All the compounds exhibit remarkable photostability under blue-light irradiation and are emissive (605 < λem < 720 nm), with the Ru(II) derivatives displaying higher photoluminescence quantum yields and longer excited state lifetimes. The Ir PSs display pKa values between 5.9 and 7.9, whereas their Ru counterparts are less acidic (pKa > 9.3). The presence of the deprotonated form in the Ir-PSs favours the generation of reactive oxygen species (ROS) since, according to theoretical calculations, it features a low-lying ligand-centered triplet excited state (T1 = 3LC) with a long lifetime. All compounds have demonstrated anticancer activity. Ir(III) complexes 1-3 exhibit the highest cytotoxicity in dark conditions, comparable to cisplatin. Their activity is notably enhanced by blue-light irradiation, resulting in nanomolar IC50 values and phototoxicity indexes (PIs) between 70 and 201 in different cancer cell lines. The Ir(III) PSs are also activated by green (with PI between 16 and 19.2) and red light in the case of complex 3 (PI = 8.5). Their antitumor efficacy is confirmed by clonogenic assays and using spheroid models. The Ir(III) complexes rapidly enter cells, accumulating in mitochondria and lysosomes. Upon photoactivation, they generate ROS, leading to mitochondrial dysfunction and lysosomal damage and ultimately cell apoptosis. Additionally, they inhibit cancer cell migration, a crucial step in metastasis. In contrast, Ru(II) complex 6 exhibits moderate mitochondrial activity. Overall, Ir(III) complexes 1-3 show potential for selective light-controlled cancer treatment, providing an alternative mechanism to chemotherapy and the ability to inhibit lethal cancer cell dissemination.

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.

Conjugation of a Ru(II) arene complex to neomycin or to guanidinoneomycin leads to compounds with differential cytotoxicities and accumulation between cancer and normal cells.

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η(6)-p-cym)RuCl(PPh3)2](+), allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 µM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 µM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 µM in DU-145 and IC50 = 11.33 µM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 ≫ 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 µM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

Study of the biological activity of photoactive bipyridyl-Ru(II) complexes containing 1,3,5-triaza-7-phosphaadamantane (PTA).

The water-soluble ruthenium complex cis-[Ru(dcbpyH)2(PTAH)2]Cl2·3H2O (1) (dcbpy = 4,4'-dicarboxy-2,2'-bipyridine; PTA = 1,3,5-triaza-7-phosphaadamantane) has been synthesized and characterised by NMR, IR spectroscopy, elemental analysis, and single-crystal X-ray diffraction. The optical properties of 1 were studied, including photoactivation under visible light, as well as its biological properties, together with those of the previously published Ru complexes cis-[Ru(bpy)2(PTA)2]Cl2 (2), trans-[Ru(bpy)2(PTA)2](CF3SO3)2 (3) and cis-[Ru(bpy)2(H2O)(PTA)](CF3SO3)2 (4) (bpy = 2,2'-bipyridine). Anticancer activities of the complexes against human lung (A549), cervical (HeLa) and prostate (PC3) carcinoma cells were evaluated under dark conditions and upon photoactivation with visible light. None of the complexes exhibited cytotoxic activity in the absence of light irradiation (IC50 > 100 µM). However, after photoactivation, the cytotoxicity of complexes 1, 2 and 3 against the three cell lines markedly increased, resulting in IC50 values between 25.3 µM and 9.3 µM. Notably, these complexes did not show toxicity against red blood cells. These findings show the potential of complexes 1, 2 and, particularly, 3 for selective and controlled cancer photochemotherapy. The reactivity of the Ru complexes against DNA under UV-Vis irradiation was studied by analysing plasmid mobility. Experimental data shows that 4 unfolds supercoiled DNA (SC DNA) both in the dark and under visible irradiation, while 1 and 3 are only active under light, being 2 inactive in either case. The unfolding activities of complexes 3 and 4 were dependent on the air present in the reaction. The measured intracellular levels of reactive oxygen species (ROS) upon irradiation with complexes 1, 2 and 3 suggest that their mechanism of action is related to oxidative stress.

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.

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.

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.

Toward Angiogenesis Inhibitors Based on the Conjugation of Organometallic Platinum(II) Complexes to RGD Peptides.

A novel conjugate between a cyclometalated platinum(II) complex with dual antiangiogenic and antitumor activity and a cyclic peptide containing the RGD sequence (-Arg-Gly-Asp-) has been synthesized by combining solid- and solution-phase methodologies. Although peptide conjugation rendered a non-cytotoxic compound in all tested tumor cell lines (± αV ß3 and αV ß5 integrin receptors), the antiangiogenic activity of the Pt-c(RGDfK) conjugate in human umbilical vein endothelial cells at sub-cytotoxic concentrations opens the way to the design of a novel class of angiogenesis inhibitors through conjugation of metallodrugs with high antiangiogenic activity to cyclic RGD-containing peptides or peptidomimetic analogues.