Efficient cytosolic delivery of luminescent lanthanide bioprobes in live cells for two-photon microscopy.

Lanthanide(iii) (Ln3+) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb3+ and Eu3+ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln3+ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln3+ complex. The cytotoxicity of these three probes was evaluated and the IC50 ranged from 7 µM to >50 µM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.

Luminescent Peptide/Lanthanide(III) Complex Conjugates with Push-Pull Antennas: Application to One- and Two-Photon Microscopy Imaging.

Lanthanide(III) (Ln3+) complexes feature desirable luminescence properties for cell microscopy imaging, but cytosolic delivery of Ln3+ complexes and their use for 2P imaging of live cells are challenging. In this article, we describe the synthesis and spectroscopic characterizations of a series of Ln3+ complexes based on two ligands, L1 and L2, featuring extended picolinate push-pull antennas for longer wavelength absorption and 2P absorption properties as well as a free carboxylate function for conjugation to peptides. Several cell penetrating peptide/Ln3+ complex conjugates were then prepared with the most interesting luminescent complexes, Tb(L1) and Eu(L2), and with two cell penetrating peptides (CPPs), ZF5.3 and TP2. A spectroscopic analysis demonstrates that the luminescence properties of the complexes are not affected by conjugation to the peptide. The conjugates were evaluated for one-photon (1P) time-gated microscopy imaging, which suppresses biological background fluorescence, and 2P confocal microscopy. Whereas TP2-based conjugates were unable to enter cells, successful 1P and 2P imaging was performed with ZF5.3[Tb(L1)]. 2P confocal imaging suggests proper internalization and cytosolic delivery as expected for this CPP. Noteworthy, 2P confocal microscopy also allowed characterization of the luminescence properties of the complex (spectrum, lifetime) within the cell, opening the way to functional luminescent probes for 2P confocal imaging of live cells.

Symmetric CEST-active lanthanide complexes for redox monitoring.

Two symmetric ligands harbouring two TEMPO radicals and two functionalized acetamide arms (R = OMe (L1), CF3 (L2)) were prepared and chelated to lanthanide ions (EuIII, YbIII for both L1 and L2, DyIII for L1). Luminescence measurements on the europium complexes support the coordination of a single water molecule. The TEMPO arms are magnetically interacting in L1 (and its complexes) but not in L2. The TEMPO moieties can be reversibly oxidized into an oxoammonium (0.33-0.36 V vs. Fc+/Fc) or reduced into a hydroxylamine (ill-defined redox wave, reduction by ascorbate), which are both diamagnetic. The europium complexes [Eu(L1)]3+ and [Eu(L2)]3+ in their hydroxylamine form exhibit a temperature dependent CEST effect, which is maximal at 25 °C (30%) and 37 °C (12%), respectively. The CEST activity is dramatically reduced in the corresponding nitroxide forms due to the paramagnetism of the ligand. The europium complexes show no cytotoxicity against M21 cell lines over long incubation times (72 h) at high concentration (40 µM).

Lanthanide Complexes (GdIII and EuIII ) Based on a DOTA-TEMPO Platform for Redox Monitoring via Relaxivity.

Three lanthanide complexes (Ln=Gd, Eu) based on a DO3 A ([Ln(L1 )]) or DO2 A ([Ln(L2-3 )]+ ) platform appended by a redox active TEMPO-based arm were prepared. Complex [Ln(L2 )]+ shows an alkyne arm, offering the possibility of postfunctionalization by click reaction to yield [Ln(L3 )]+ . The complexes demonstrate a redox response whereby the hydroxylamine, nitroxide and oxoammonium forms of the arm can be obtained in turn. Luminescence measurements on the europium complexes support an octadentate (L1 , L3 ) or heptadentate (L2 ) chelation by the ligand, with one water molecule in the inner coordination sphere. The relaxivity was determined from 20 kHz to 30 MHz by fast-field cycling NMR. The three GdIII complexes under their hydroxylamine form [Gd(L1 )] and [Gd(L2-3 )]+ show r1 values of 7.0, 5.1 and 5.0 mM-1 s-1 (30 KHz), which increase to 8.8, 5.5 and 6.1 mM-1 s-1 in the nitroxide form. The radical complexes are not toxic against M21 cell lines, at least up to 40 µM. By using EPR spectroscopy we establish that they do not penetrate the cells with the exception of [Eu(L2 )]+ .

Mechano-Bactericidal Titanium Surfaces for Bone Tissue Engineering.

Despite advances in the development of bone substitutes and strict aseptic procedures, the majority of failures in bone grafting surgery are related to nosocomial infections. Development of biomaterials combining both osteogenic and antibiotic activity is, therefore, a crucial public health issue. Herein, two types of intrinsically bactericidal titanium supports were fabricated by using commercially scalable techniques: plasma etching or hydrothermal treatment, which display two separate mechanisms of mechano-bactericidal action. Hydrothermal etching produces a randomly nanostructured surface with sharp nanosheet protrusions killing bacteria via cutting of the cell membrane, whereas plasma etching of titanium produces a microscale two-tier hierarchical topography that both reduce bacterial attachment and rupture those bacteria that encounter the surface. The adhesion, growth, and proliferation of human adipose-derived stem cells (hASCs) on the two mechano-bactericidal topographies were assessed. Both types of supports allowed the growth and proliferation of the hASCs in the same manner and cells retained their stemness and osteogenic potential. Furthermore, these supports induced osteogenic differentiation of hASCs without the need of differentiation factors, demonstrating their osteoinductive properties. This study proves that these innovative mechano-bactericidal titanium surfaces with both regenerative and bactericidal properties are a promising solution to improve the success rate of reconstructive surgery.

Prognostic Value of Soluble Death Receptor Ligands in Patients with Transitional Cell Carcinoma of Bladder.

BACKGROUND: The activation of Fas/Fas ligand (FasL) and DR4-DR5/tumor necrosis factor-related-apoptosis-inducing ligand (TRAIL) pathways in cancer cells triggers apoptosis. The objective of this study was to investigate the prognostic value of soluble FasL (sFasL) and soluble (sTRAIL) in the serum of patients with bladder cancer. METHODS: The sFasL and sTRAIL levels in the sera of patients with bladder cancer or healthy donors were determined using the enzyme-linked immunosorbent assay. Micro-culture tetrazolium viability assay and Western blot were used to analyze cell cytotoxicity and death receptors protein expression respectively. RESULTS: Whether no difference in sTRAIL levels was seen between patients and controls, the level of sFasL was higher in patients than that in healthy donors. According to, sFasL level was the highest in the serum of patients with superficial stage or low- and medium-grade cancer. Moreover, sFasL in patients with superficial noninvasive bladder tumors or low- and medium-grade cancers was higher than that in patients with invasive carcinomas and high-grade cancers. Patients with high levels of sFasL survive longer than those with low levels, probably related to the cytotoxic potential of FasL preserved in its soluble form. CONCLUSION: The data suggest that monitoring the level of sFasL and its cytotoxic activity could be a prognostic marker in the follow-up of patients with bladder cancer.

Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Anti-proliferative and nuclease activities.

A series of tripodal ligands based on the 2-tert-butyl-4-R-6-phenol was synthesized, where R=aldehyde (HL1), R=putrescine-pyrene (HL2) and R=putrescine (HL3). A dinucleating ligand wherein a putrescine group connects two tripodal moieties was also prepared (H2L4). The corresponding copper complexes (1, 2, 3, and 4, respectively) were prepared and characterized. We determined the phenol's pKas in the range 2.47-3.93. The DNA binding constants were determined at 6×106, 5.5×105 and 2.7×106 for 2, 3 and 4, respectively. The complexes display a metal-centered reduction wave at Epc,red=-0.45 to -0.5V vs. saturated calomel electrode, as well as a ligand-centered oxidation wave above 0.57V at pH7. In the presence of ascorbate they promote an efficient cleavage of DNA, with for example a concentration required to cleave 50% of supercoiled DNA of 1.7µM for 2. The nuclease activity is affected by the nature of the R group: putrescine-pyrene≈bis-ligating>putrescine>aldehyde. The species responsible for strand scission is the hydroxyl radical. The cytotoxicity of the complexes was evaluated on bladder cancer cell lines sensitive or resistant to cis-platin. The IC50 of complexes 2 and 4 span over a short range (1.3-2µM) for the two cell lines. They are lower than those of the other complexes (3.1-9.7µM) and cis-platin. The most active compounds block the cell cycle at the G0/1 phase and promote apoptosis.

Identification of pyrrolopyrimidine derivative PP-13 as a novel microtubule-destabilizing agent with promising anticancer properties.

Despite the emergence of targeted therapies and immunotherapy, chemotherapy remains the gold-standard for the treatment of most patients with solid malignancies. Spindle poisons that interfere with microtubule dynamics are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of chemoresistance highlight the need for identifying alternative agents. We performed a high throughput cell-based screening and selected a pyrrolopyrimidine molecule (named PP-13). In the present study, we evaluated its anticancer properties in vitro and in vivo. We showed that PP-13 exerted cytotoxic effects on various cancer cells, including those resistant to current targeted therapies and chemotherapies. PP-13 induced a transient mitotic blockade by interfering with both mitotic spindle organization and microtubule dynamics and finally led to mitotic slippage, aneuploidy and direct apoptotic death. PP-13 was identified as a microtubule-targeting agent that binds directly to the colchicine site in ß-tubulin. Interestingly, PP-13 overcame the multidrug-resistant cancer cell phenotype and significantly reduced tumour growth and metastatic invasiveness without any noticeable toxicity for the chicken embryo in vivo. Overall, PP-13 appears to be a novel synthetic microtubule inhibitor with interesting anticancer properties and could be further investigated as a potent alternative for the management of malignancies including chemoresistant ones.

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

RATIONALE: Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. METHODS: Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. RESULTS: Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. CONCLUSION: This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society.

Cytotoxicity of chitosans with different acetylation degrees and molecular weights on bladder carcinoma cells.

The purpose of this research was to evaluate the cytotoxicity of chitosans with different degrees of acetylation (DA) and molecular weights (MW), as well as the effect of their positive ionic charges controlled by pH on bladder carcinoma cells (RT112 and RT112cp) using the tetrazolium salt colorimetric (MTT) assay. Our data showed that all chitosan samples were cytotoxic on RT112 and RT112cp cells with a higher cytotoxicity obtained at lower pH. Further, it was found that the toxicity increased with increasing DA. However, no significant difference in cytotoxicity between chitosans with different molecular weights was observed. Annexin V-FITC staining test was then used to study and quantify the induction of apoptosis. Data shows that chitosans induce apoptosis of RT112 and RT112cp cells with the same dependence with DA.

IPP51, a chalcone acting as a microtubule inhibitor with in vivo antitumor activity against bladder carcinoma.

We previously identified 1-(2,4-dimethoxyphenyl)-3-(1-methylindolyl) propenone (IPP51), a new chalcone derivative that is capable of inducing prometaphase arrest and subsequent apoptosis of bladder cancer cells. Here, we demonstrate that IPP51 selectively inhibits proliferation of tumor-derived cells versus normal non-tumor cells. IPP51 interfered with spindle formation and mitotic chromosome alignment. Accumulation of cyclin B1 and mitotic checkpoint proteins Bub1 and BubR1 on chromosomes in IPP51 treated cells indicated the activation of spindle-assembly checkpoint, which is consistent with the mitotic arrest. The antimitotic actions of other chalcones are often associated with microtubule disruption. Indeed, IPP51 inhibited tubulin polymerization in an in vitro assay with purified tubulin. In cells, IPP51 induced an increase in soluble tubulin. Furthermore, IPP51 inhibited in vitro capillary-like tube formation by endothelial cells, indicating that it has anti-angiogenic activity. Molecular docking showed that the indol group of IPP51 can be accommodated in the colchicine binding site of tubulin. This characteristic was confirmed by an in vitro competition assay demonstrating that IPP51 can compete for colchicine binding to soluble tubulin. Finally, in a human bladder xenograft mouse model, IPP51 inhibited tumor growth without signs of toxicity. Altogether, these findings suggest that IPP51 is an attractive new microtubule-targeting agent with potential chemotherapeutic value.

Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan.

Chitin was recovered through enzymatic deproteinization of the shrimp processing by-products. Different microbial and fish viscera proteases were tested for their deproteinization efficiency. High levels of protein removal of about 77±3% and 78±2% were recorded using Bacillus mojavensis A21 and Balistes capriscus proteases, respectively, after 3h of hydrolysis at 45°C using an enzyme/substrate ratio of 20U/mg. Therefore, these two crude proteases were used separately for chitin extraction and then chitosan preparation by N-deacetylation. Chitin and chitosan samples were then characterized by 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and compared to samples prepared through chemical deproteinization. All chitins and chitosans showed identical spectra. Chitosans prepared through enzymatic deproteinization have practically the same acetylation degree but higher molecular weights compared to that obtained through chemical process. Antimicobial, antioxidant and antitumoral activitities of chitosan-M obtained by treatment with A21 proteases and chitosan-C obtained by alkaline treatment were investigated. Results showed that both chitosans inhibited the growth of most Gram-negative, Gram-positive bacteria and fungi tested. Furthermore, both chitosans exhibited antioxidant and antitumor activities which was dependent on the molecular weight.

AG11, a novel dichloroflavanone derivative with anti-mitotic activity towards human bladder cancer cells.

BACKGROUND: New chemotherapy drugs should be investigated to improve survival of patients with advanced bladder cancer. Here, we report the synthesis and evaluation of AG11, a new flavanone derivative obtained through cyclization of its chalcone precursor CB11. MATERIALS AND METHODS: The effect of AG11 on cell viability was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay and apoptotic cell death was analyzed by flow cytometry. Finally, the effect of AG11 on tubulin polymerization in vitro and microtubule distribution across the cells was investigated. RESULTS: AG11 was found to have an IC50 (half-maximal inhibitory concentration) of 4.6 µM and its inhibitory effect on RT4 cells proliferation is associated with a cell-cycle arrest in G2+M phases followed by apoptosis after a 48 h treatment. AG11 prevented polymerization of purified tubulin in a concentration-dependent manner in vitro and disrupted mitotic spindle formation in cells. CONCLUSION: AG11 appears to be an attractive scaffold for further development of a structurally simpler new anti-microtubule agents.

Nuclease and anti-proliferative activities of copper(II) complexes of N3O tripodal ligands involving a sterically hindered phenolate.

Copper(II) complexes 1(2+)-6 of a series of tripodal ligands involving a N3O donor set, namely 2-[(bis-pyridin-2-ylmethyl-amino)-methyl]-4-methoxy-phenol (1L), 2-tert-butyl-4-methoxy-6-[bis-pyridin-2-ylmethyl-amino)-methyl]-phenol (2L), 2-tert-butyl-4-methoxy-6-{[(2-pyridin-2-yl-ethyl)-pyridin-2-ylmethyl-amino]-methyl}-phenol (3L), 2-tert-butyl-4-methoxy-6-{[(6-methyl-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-amino]-methyl}-phenol (4L), 2-tert-butyl-4-fluoro-6-{[(6-methyl-pyridin-2-ylmethyl)-pyridin-2-ylmethyl-amino]-methyl}-phenol (5L) and 2-tert-butyl-4-methoxy-6-{bis[(6-methyl-pyridin-2-ylmethyl)-amino]-methyl}-phenol (6L), respectively, were synthesized. Complexes 1(2+), 3(+) and 4(+) were structurally characterized by X-ray diffraction. The structure of 1(2+) is dimeric, with an essentially trigonal bipyramidal geometry around the copper(II) ions and two bridging deprotonated phenolate moieties. The mononuclear complexes 3(+) and 4(+) contain a square pyramidal copper ion, coordinated in axial position by the phenol moiety. In the water-DMF (90 : 10) mixture at pH 7.3 all the copper(II) complexes are mononuclear, mainly under their phenolate neutral form (except 3(+)), with a coordinated solvent molecule. The DNA cleavage activity of the complexes was tested towards the ϕX174 DNA plasmid. In the absence of an exogenous agent 1(2+) does not show any cleavage activity, 2(+) and 3(+) are moderately active, while 4(+), 5(+) and 6(+) exhibit a high nuclease activity. Experiments in the presence of various scavengers reveal that reactive oxygen species (ROS) are not involved in the strand scission mechanism. The cytotoxicity of the complexes was evaluated on bladder cancer cell lines sensitive or resistant to cisplatin. The IC50 values of the complexes 2(+), 4(+), 5(+) and 6(+) are lower than that of cisplatin (range from 6.3 to 3.1 µM against 9.1 µM for cisplatin). Furthermore, complexes 2(+), 4(+), 5(+) and 6(+) are able to circumvent cisplatin cellular resistance.

Investigation of chalcones as selective inhibitors of the breast cancer resistance protein: critical role of methoxylation in both inhibition potency and cytotoxicity.

ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC(50) values below 0.5 µM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6'-hydroxyl-2',4'-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.

Flavones inhibit the proliferation of human tumor cancer cell lines by inducing apoptosis.

Dietary flavonoids have been shown to exert specific cytotoxicity toward some cancer cells, but the precise molecular mechanisms are still not completely understood. In this study, cytotoxic effects of flavones (apigenin and luteolin) on two different cancer cell lines, including human chronic myelogenous erythroleukaemia (K562) and bladder carcinoma (RT112), were determined, and the molecular mechanisms responsible for their cytotoxic effects were studied. The results of an MTT assay showed that luteolin and apigenin were able to induce cytotoxicity in K562 and RT112 cells in a dose- and time-dependent manner. The cytotoxic potency of luteolin was higher than that of apigenin. Flow-cytometry and DNA-fragmentation analysis indicated that the cytotoxicity induced by luteolin and apigenin was mainly due to apoptosis, with minor cell-cycle perturbations. This apoptotic response was characterized by an increase of the sub-G1 fraction of treated cells, poly(ADP-ribose) polymerase proteolysis, typical ladder of DNA fragmentation, and Annexin V-positive cells. In conclusion, luteolin and apigenin exert cytotoxic effects in different cancer cell lines in which apoptosis plays an important role. Thus, flavones could be considered as potential chemotherapeutic agents.

Structural requirement of arylindolylpropenones as anti-bladder carcinoma cells agents.

Chalcones have been identified as interesting compounds with cytotoxicity, anti-inflammatory and antioxidant properties. In the present study, we report the synthesis and evaluation of new 1-(N-methylindolyl)-3-phenylpropenones as anti-cancer agents acting on bladder carcinoma cell line. Among the 15 investigated molecules, three of them inhibit the growth of bladder cancer cells with IC(50) values less than 4 µM after 48 h of treatment. To investigate their mode of action, cell cycle analyses were performed. The most active compounds induce high accumulation at the G2+M phase as assessed by flow cytometry. The structure-activity relationship drawn from the present study highlights the importance of the substitution pattern of the phenyl ring and provides valuable information for further development of this class of compounds as novel anti-cancer chemotherapeutic agents.