L-659,989: A useful probe in the detection of multiple conformational states of PAF receptors

L-659,989 is a potent, specific and competitive plateletactivating factor (PAF) receptor antagonist. The 2,5-tritium labeled L-659,989, similar to [³H]PAF, specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (K_D) of 1.60 (±0.20) nM in 10 mM MgCl₂. However, guanosine 5′-triphosphate (GTP) and several cations affect the specific binding of [³H]PAF and of [³H]L-659,989 to rabbit platelet membranes in different ways. K⁺, Mg²⁺, Ca²⁺ and Mn²⁺ potentiate the specific binding of both ligands. Na⁺ and Li⁺ inhibit the specific [³H]PAF binding, but enhance the binding of [³H]L-659,989; GTP reduces the [³H]PAF binding but has no effect on the binding of [³H]-L-659,989. Ni²⁺ inhibits the [³H]L-659-989 binding, but has no effect on the binding of [³H]PAF. In the presence of 150 mM NaCl, [³H]L-659,989 exhibits identical K_D and detectable binding sites (B_max) values as those in the presence of 10 mM MgCl₂, while K _d And B_max values of [³H]PAF are dramatically reduced in the presence of 150 mM NaCl compared to those in 10 mM MgCl₂. These results suggest the existence of multiple conformational states of the PAF specific receptor and that PAF and L-659,989 bind differently to those states. In the presence of 150 mM NaCl and 1 mM GTP, receptors appear to exist in a single conformational state with an equilibrium dissociation constant (K_B) of 0.93 μM for PAF as derived from the Schild plot. In isolated rabbit platelets pretreated with 10 μM ETH 227, a Na⁺-specific ionophore, the detectable [³H]PAF binding sites drop from 260 to 100 binding sites per platelet, but the binding sites for [³H]L-659,989 remain roughly the same. The Na⁺ binding sites which modulate the conformation of PAF receptors are therefore protected from extracellular Na⁺ until ionophore is added, and are probably located on the cytoplasmic side of the plasma membrane. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen,Hydrogen Peroxide,Nitric Oxide and Nitrite/Nitrate

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H₂O₂ present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.     

A Pyrazolate Osmium(VI) Nitride Exhibits Anticancer Activity through Modulating Protein Homeostasis in HepG2 Cells

Interest in the third-row transition metal osmium and its compounds as potential anticancer agents has grown in recent years. Here, we synthesized the osmium(VI) nitrido complex Na[Os^VI(N)(tpm)₂] (tpm = [5-(Thien-2-yl)-1H-pyrazol-3-yl]methanol), which exhibited a greater inhibitory effect on the cell viabilities of the cervical, ovarian, and breast cancer cell lines compared with cisplatin. Proteomics analysis revealed that Na[Os^VI(N)(tpm)₂] modulates the expression of protein-transportation-associated, DNA-metabolism-associated, and oxidative-stress-associated proteins in HepG2 cells. Perturbation of protein expression activity by the complex in cancer cells affects the functions of the mitochondria, resulting in high levels of cellular oxidative stress and low rates of cell survival. Moreover, it caused G2/M phase cell cycle arrest and caspase-mediated apoptosis of HepG2 cells. This study reveals a new high-valent osmium complex as an anticancer agent candidate modulating protein homeostasis.     

Cell‐Selective,Apoptosis‐inducing Rhodium(III) Crown Thiaether Complexes

Half‐sandwich rhodium(III) polypyridyl (pp) complexes with the metal atom capped by the facial crown thiaether 1,4,7‐trithiacyclononane [9]aneS₃ represent a promising class of apoptosis‐inducing potent cytostatic agents. The necrotic damage caused by the complexes is negligible. In vitro cytotoxicity assays with the human cancer cell lines MCF‐7 and HT‐29 and immortalized HEK‐293 cells indicate that the dicationic κ²N(imino) complexes [([9]aneS₃)RhCl(pp)]²⁺ are much more active than monocationic complexes [([9]aneS₃)RhCl₂(L)]⁺ (L=imidazole, CH₃CN). Whereas the κ²N(amino) complex [([9]aneS₃)RhCl(piperazine)]²⁺ is inactive, replacing piperazine with the structurally analogous κ²S (thiaether) ligand 1,4‐dithiane restores cytotoxicity as evidenced by IC₅₀ values in the range 8.1‐11.6 μM . Spectroscopic (CD, UV/Vis, NOESY) and viscosity measurements indicate that the active dppz complex 8 (IC₅₀ values: 4.7–8.9 μM ) exhibits strong intercalative binding towards DNA whereas the even more potent bipyrimidine complex 9 (IC₅₀ values: 0.6–1.9 μM ) causes no alteration of the duplex B conformation. Weaker intercalative binding is observed for the dpq complex 7 . A comparative annexin V–propidium iodide binding assay with lymphoma (BJAB) cells and healthy leukocytes demonstrates that the cytotoxic activity of complex 8 and particularly complex 9 is highly selective towards the malignant cells.     

Imaging of KCa3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes

The Ca²⁺ activated K⁺ channel K_Ca3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this K_Ca3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [¹⁸F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of K_Ca3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms ( 9 a ) and with a F-atom and a methoxy moiety ( 9 b ) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single K_Ca3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of K_Ca3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the K_Ca3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.     

Dinitrophenol-Hyaluronan Conjugates as Multivalent Antibody-Recruiting Glycopolymers for Targeted Cancer Immunotherapy

Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG₃-DNP]₈, showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG₃-DNP]₈ significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.     

Synthesis and Anti-Cancer Activity of New Pyrazolinyl-Indole Derivatives: Pharmacophoric Interactions and Docking Studies for Identifying New EGFR Inhibitors

Newly designed series of indole-containing pyrazole analogs, pyrazolinylindoles, were synthesized, and their structures were confirmed based on the spectral data of the ¹H NMR, ¹³C NMR, and HR-MS analyses. Preliminary anti-cancer activity testings were carried out by the National Cancer Institute, United States of America (NCI, USA). Compounds HD02, HD05, and HD12 demonstrated remarkable cytotoxic activities against nine categories of cancer types based cell line panels which included leukemia, colon, breast, melanoma, lungs, renal, prostate, CNS, and ovarian cancer cell lines. The highest cytotoxic effects were exhibited by the compounds HD02 [1-(5-(1-H-indol-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenylethanone], HD05 [1-(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenoxyethanone], and HD12 [(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone] against some of the 56 types of NCI-based cell lines in different panels. Compound HD05 showed the maximum range of cancer cell growth inhibitions against all categories of the cell lines in all nine panels. On average, in comparison to the referral standard, imatinib, at a dose level of 10 µM, the HD05 showed significant activity against leukemia in the range of 78.76%, as compared to the imatinib at 9% of cancer cells’ growth inhibitions. Molecular docking simulation studies were performed in silico on the epidermal growth factor receptor (EGFR) tyrosine kinase, in order to validate the activity.     

Mechanisms of the Cytotoxic Effect of Selenium Nanoparticles in Different Human Cancer Cell Lines

In recent decades, studies on the functional features of Se nanoparticles (SeNP) have gained great popularity due to their high biocompatibility, stability, and pronounced selectivity. A large number of works prove the anticarcinogenic effect of SeNP. In this work, the molecular mechanisms regulating the cytotoxic effects of SeNP, obtained by laser ablation, were studied by the example of four human cancer cell lines: A-172 (glioblastoma), Caco-2, (colorectal adenocarcinoma), DU-145 (prostate carcinoma), MCF-7 (breast adenocarcinoma). It was found that SeNP had different concentration-dependent effects on cancer cells of the four studied human lines. SeNP at concentrations of less than 1 μg/mL had no cytotoxic effect on the studied cancer cells, with the exception of the A-172 cell line, for which 0.5 μg/mL SeNP was the minimum concentration affecting its metabolic activity. It was shown that SeNP concentration-dependently caused cancer cell apoptosis, but not necrosis. In addition, it was found that SeNP enhanced the expression of pro-apoptotic genes in almost all cancer cell lines, with the exception of Caco-2 and activated various pathways of adaptive and pro-apoptotic signaling pathways of UPR. Different effects of SeNP on the expression of ER-resident selenoproteins and selenium-containing glutathione peroxidases and thioredoxin reductases, depending on the cell line, were established. In addition, SeNP triggered Ca²⁺ signals in all investigated cancer cell lines. Different sensitivity of cancer cell lines to SeNP can determine the induction of the process of apoptosis in them through regulation of the Ca²⁺ signaling system, mechanisms of ER stress, and activation of various expression patterns of genes encoding pro-apoptotic proteins.     

Design,Synthesis, Radiosynthesis and Biological Evaluation of Fenretinide Analogues as Anticancer and Metabolic Syndrome-Preventive Agents

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies showed its promising therapeutic potential for preventing metabolic syndrome. Several biological targets are involved in 4-HPR's activity, leading to the potential use of this molecule for treating different pathologies. However, although 4-HPR displays quite well-understood multitarget promiscuity with regards to pharmacology, interpreting its precise physiological role remains challenging. In addition, despite promising results in vitro, the clinical efficacy of 4-HPR as a chemotherapeutic agent has not been satisfactory so far. Herein, we describe the preparation of a library of 4-HPR analogues, followed by the biological evaluation of their anti-cancer and anti-obesity/diabetic properties. The click-type analogue 3 b showed good capacity to reduce the amount of lipid accumulation in 3T3-L1 adipocytes during differentiation. Furthermore, it showed an IC₅₀ of 0.53±0.8 μM in cell viability tests on breast cancer cell line MCF-7, together with a good selectivity (SI=121) over noncancerous HEK293 cells. Thus, 3 b was selected as a potential PET tracer to study retinoids in vivo, and the radiosynthesis of [¹⁸F] 3b was successfully developed. Unfortunately, the stability of [¹⁸F] 3b turned out to be insufficient to pursue imaging studies.     

Polyoxometalate Nanoparticles as a Potential Glioblastoma Therapeutic via Lipid-Mediated Cell Death

Polyoxometalate nanoparticles (POMs) are a class of compounds made up of multiple transition metals linked together using oxygen atoms. POMs commonly include group 6 transition metals, with two of the most common forms using molybdenum and tungsten. POMs are suggested to exhibit antimicrobial effects. In this study, we developed two POM preparations to study anti-cancer activity. We found that Mo-POM (NH₄)Mo₇O₂₄) and W-POM (H₃PW₁₂O₄₀) have anti-cancer effects on glioblastoma cells. Both POMs induced morphological changes marked by membrane swelling and the presence of multinucleated cells that may indicate apoptosis induction along with impaired cell division. We also observed significant increases in lipid oxidation events, suggesting that POMs are redox-active and can catalyze detrimental oxidation events in glioblastoma cells. Here, we present preliminary indications that molybdenum polyoxometalate nanoparticles may act like ferrous iron to catalyze the oxidation of phospholipids. These preliminary results suggest that Mo-POMs (NH₄)Mo₇O₂₄) and W-POMs (H₃PW₁₂O₄₀) may warrant further investigation into their utility as adjunct cancer therapies.     

Interaction of the σ2 Receptor Ligand PB28 with the Human Nucleosome: Computational and Experimental Probes of Interaction with the H2A/H2B Dimer

Sigma‐2 (σ₂) binding sites are an emerging target for anti‐neoplastic agents due to the strong apoptotic effect exhibited by σ₂ agonists in vitro and the overexpression of these sites in tumor cells. Nonetheless, no σ₂ receptor protein has been identified. Affinity chromatography using the high‐affinity σ₂ ligand PB28 and human SK‐N‐SH neuroblastoma cells was previously utilized to identify σ₂ ligand binding proteins, specifically histones H1, H2A, H2B, and H3.3a. To rationalize this finding, homology modeling and automated docking studies were employed to probe intermolecular interactions between PB28 and human nucleosomal proteins. These studies predicted interaction of PB28 with the H2A/H2B dimer at a series of sites previously found to be implicated in chromatin compaction and nucleosomal assembly. To experimentally verify this prediction, a competitive binding assay was performed on the reconstituted H2A/H2B dimer using [³H]PB28 as radioligand, and an IC₅₀ value of 0.50 nM was determined for PB28 binding. In addition, [³H]PB28 was found to accumulate with up to a fivefold excess in nuclear fractions over cytosolic fractions of SK‐N‐SH and MCF7 cells, indicating that PB28 is capable of entering the nucleus to interact with histone proteins. In conjunction with computational results, these data suggest that PB28 may exert its cytotoxic effect through direct interaction with nuclear material.     

Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2

A cytosolic 84 kDa Group VIA phospholipase A₂ (iPLA₂β) that does not require Ca²⁺ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β-cells, and other sources. Proposed iPLA₂β functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (IPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet β-cells. To further examine iPLA₂β functions in β-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA₂β activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPIA₂β cDNA. The iPLA₂β-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [³H] choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA₂β-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [³H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA₂β. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA₂β-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA₂β-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA₂β plays a signaling role in β-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.     

Novel A-Ring Chalcone Derivatives of Oleanolic and Ursolic Amides with Anti-Proliferative Effect Mediated through ROS-Triggered Apoptosis

A series of A-ring modified oleanolic and ursolic acid derivatives including C28 amides (3-oxo-C2-nicotinoylidene/furfurylidene, 3β-hydroxy-C2-nicotinoylidene, 3β-nicotinoyloxy-, 2-cyano-3,4-seco-4(23)-ene, indolo-, lactame and azepane) were synthesized and screened for their cytotoxic activity against the NCI-60 cancer cell line panel. The results of the first assay of thirty-two tested compounds showed that eleven derivatives exhibited cytotoxicity against cancer cells, and six of them were selected for complete dose–response studies. A systematic study of local SARs has been carried out by comparative analysis of potency distributions and similarity relationships among the synthesized compounds using network-like similarity graphs. Among the oleanane type triterpenoids, C2-[4-pyridinylidene]-oleanonic C28-morpholinyl amide exhibited sub-micromolar potencies against 15 different tumor cell lines and revealed particular selectivity for non-small cell lung cancer (HOP-92) with a GI₅₀ value of 0.0347 μM. On the other hand, superior results were observed for C2-[3-pyridinylidene]-ursonic N-methyl-piperazinyl amide 29, which exhibited a broad-spectrum inhibition activity with GI₅₀ < 1 μM against 33 tumor cell lines and <2 μM against all 60 cell lines. This compound has been further evaluated for cell cycle analysis to decipher the mechanism of action. The data indicate that compound 29 could exhibit both cytostatic and cytotoxic activity, depending on the cell line evaluated. The cytostatic activity appears to be determined by induction of the cell cycle arrest at the S (MCF-7, SH-SY5Y cells) or G₀/G₁ phases (A549 cells), whereas cytotoxicity of the compound against normal cells is nonspecific and arises from apoptosis without significant alterations in cell cycle distribution (HEK293 cells). Our results suggest that the antiproliferative effect of compound 29 is mediated through ROS-triggered apoptosis that involves mitochondrial membrane potential depolarization and caspase activation.     

Synchrotron-Based Fourier-Transform Infrared Micro-Spectroscopy (SR-FTIRM) Fingerprint of the Small Anionic Molecule Cobaltabis(dicarbollide) Uptake in Glioma Stem Cells

The anionic cobaltabis (dicarbollide) [3,3′-Co(1,2-C₂B₉H₁₁)₂]⁻, [o-COSAN]⁻, is the most studied icosahedral metallacarborane. The sodium salts of [o-COSAN]⁻ could be an ideal candidate for the anti-cancer treatment Boron Neutron Capture Therapy (BNCT) as it possesses the ability to readily cross biological membranes thereby producing cell cycle arrest in cancer cells. BNCT is a cancer therapy based on the potential of ¹⁰B atoms to produce α particles that cross tissues in which the ¹⁰B is accumulated without damaging the surrounding healthy tissues, after being irradiated with low energy thermal neutrons. Since Na[o-COSAN] displays a strong and characteristic ν(B-H) frequency in the infrared range 2.600–2.500 cm⁻¹, we studied the uptake of Na[o-COSAN] followed by its interaction with biomolecules and its cellular biodistribution in two different glioma initiating cells (GICs), mesenchymal and proneural respectively, by using Synchrotron Radiation-Fourier Transform Infrared (FTIR) micro-spectroscopy (SR-FTIRM) facilities at the MIRAS Beamline of ALBA synchrotron light source. The spectroscopic data analysis from the bands in the regions of DNA, proteins, and lipids permitted to suggest that after its cellular uptake, Na[o-COSAN] strongly interacts with DNA strings, modifies proteins secondary structure and also leads to lipid saturation. The mapping suggests the nuclear localization of [o-COSAN]⁻, which according to reported Monte Carlo simulations may result in a more efficient cell-killing effect compared to that in a uniform distribution within the entire cell. In conclusion, we show pieces of evidence that at low doses, [o-COSAN]⁻ translocates GIC cells’ membranes and it alters the physiology of the cells, suggesting that Na[o-COSAN] is a promising agent to BNCT for glioblastoma cells.     

Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A₁R (CPA, CCPA) or A_2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A₁R (DPCPX) or A_2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [³H]GUO binding became saturable at 100–300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [³H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [³H]GUO binding, but the sum of the two effects was able to displace [³H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A₁R and A_2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.     

Flexible Etherified and Esterified Triphenylethylene Derivatives and Their Evaluation on ER-positive and Triple-Negative Breast Cancer Cell Lines

Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) with potential clinical benefits for all stages of breast cancer. TAM is primarily metabolized to more potent metabolites via polymorphic CYP2D6. This affects the clinical outcome of TAM treatment. Herein we report novel TAM analogues that can avoid metabolism via CYP2D6. The novel analogues bear a flexible skeleton. Compounds have either an ester group on ring C or homodiaminoalkoxy groups on rings B and C . Compound 6 (E/Z-4-[1-[4-(2-diethylaminoethoxy)phenyl]-3-(4-methoxyphenyl)-2-methyl[propenyl]phenol) was found to be ten-fold more potent than TAM on MCF-7 cells (GI₅₀=0.15 μM). It showed fivefold greater inhibitory activity on MDA-MB-231 cells than TAM (GI₅₀=1.71 μM). Compound 13 (4-{3,3-bis-[4-(3-dimethylaminopropoxy)phenyl]-2-methylallyl}methoxybenzene) was the most potent among the homodiaminoalkoxy derivatives (GI₅₀=0.44) on both MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, the COMPARE algorithm suggested that it has different molecular targets from those of some other reported anticancer drugs.     

Increased Lipophilicity of Halogenated Ruthenium(II) Polypyridyl Complexes Leads to Decreased Phototoxicity in vitro when Used as Photosensitizers for Photodynamic Therapy

In the fight against cancer, photodynamic therapy is generating great interest thanks to its ability to selectively kill cancer cells without harming healthy tissues. In this field, ruthenium(II) polypyridyl complexes, and more specifically, complexes with dipyrido[3,2-a:2’,3’-c]phenazine (dppz) as a ligand are of particular interest due to their DNA-binding and photocleaving properties. However, ruthenium(II) polypyridyl complexes can sometimes suffer from low lipophilicity, which hampers cellular internalisation through passive diffusion. In this study, four new [Ru(dppz-X₂)₃]²⁺ complexes (X=H, F, Cl, Br, I) were synthesized and their lipophilicity (logP), cytotoxicity and phototoxicity on cancerous and noncancerous cell lines were assessed. This study shows that, counterintuitively, the phototoxicity of these complexes decreases as their lipophilicity increases; this could be due solely to the atomic radius of the halogen substituents.     

New Organometallic Tetraphenylethylene⋅Iridium(III) Complexes with Antineoplastic Activity

Iridium(III) complexes have attracted more and more attention in the past few years because of their potential antineoplastic activity. In this study, four Ir^III complexes of the types [(η⁵-Cp^x)Ir(N^N)Cl]PF₆ (complexes 1 and 2 ) and [Ir(Phpy)₂(N^N)]PF₆ (complexes 3 and 4 ) have been synthesized and characterized. They exhibit potential antineoplastic activity towards A549 cells, especially in the case of complex 1 [IC₅₀=(3.56±0.5) μm ], which was nearly six times as effective as cisplatin [(21.31±1.7) μm ]. Additionally, these complexes show some selectivity towards cancer cells over normal cells. They could be transported by serum albumin (binding constants were changed from 0.37×10⁵ to 81.71×10⁵ m ⁻¹). Ir^III complexes 1 and 2 could catalyze the transformation of nicotinamide adenine dinucleotide reduced form (NADH) into NAD⁺ (turnover numbers 43.2, 11.9] and induce the accumulation of reactive oxygen species, thus confirming their antineoplastic mechanism of oxidation, whereas the cyclometalated complexes 3 and 4 were able to target the lysosome [Pearson co-localization coefficient (PCC)=0.73], cause lysosomal damage, and induce apoptosis. Understanding the mechanism of action would help further structure–activity optimization on these Ir^III complexes as emerging cancer therapeutics.     

Radiolabeled 6-(2, 3-Dichlorophenyl)-N4-methylpyrimidine-2, 4-diamine (TH287): A Potential Radiotracer for Measuring and Imaging MTH1

MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [³H]TH287 to MTH1 was evaluated in live glioblastoma cells (U251MG) through saturation and competitive binding assays, together with in vitro enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 for in vivo microPET studies. Saturation binding assays show that [³H]TH287 has a dissociation constant (K_d) of 1.97 ± 0.18 nM, B_max of 2676 ± 122 fmol/mg protein for U251MG cells, and n_H of 0.98 ± 0.02. Competitive binding assays show that TH287 (K_i: 3.04 ± 0.14 nM) has a higher affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib (K_i: 153.90 ± 20.48 nM). In vitro enzymatic assays show that TH287 has an IC₅₀ of 2.2 nM in inhibiting MTH1 hydrolase activity and a K_i of 1.3 nM from kinetics assays, these results are consistent with our radioligand binding assays. Furthermore, MicroPET imaging shows that [¹¹C]TH287 gets into the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous system disorders.     

Optimization of methods and treatment conditions for studying effects of fatty acids on cell growth

Antiproliferative properties of molecular regulators of lipid metabolism have been increasingly studied during recent years. Discussion is ongoing concerning optimal treatment conditions and assays used for monitoring proliferation and cytotoxicity. The objective of the present work was to optimize methods and treatment conditions used for studying antiproliferative effects of fatty acids and analogs, represented by palmitic acid (PA) and the β-oxidation-restricted fatty acid analog tetradecylthioacetic acid (TTA), in rat (BT4Cn) and human (D54Mg and GaMg) glioma cell lines. Changes in [³H]thymidine incorporation preceded changes in cell number in TTA-treated glioma cell cultures, and the growth inhibition was more significantly expressed by [³H]thymidine incorporation than cell number. Addition of bovine serum albumin decreased cellular fatty acid uptake and reduced the effects of TTA and PA on [³H]thymidine incorporation. Determination of the antiproliferative effect of TTA in BT4Cn cells by MTT conversion and [³H]thymidine incorporation yielded concordant results. TTA-mediated reduction in cell number corresponded to reduction in cellular protein and total DNA content in BT4Cn cells. Reduced growth potential in TTA-treated multicellular D54Mg and GaMg spheroids supported the findings from monolayer cultures. In conclusion, cell density, treatment period, fatty acid administration, and methods for growth determination may profoundly influence the outcome of cell growth experiments. Thus, experimental conditions should be carefully controlled when performing cell growth experiments, and effects on cell growth should preferably be confirmed by different methods. Karl Johan Tronstad and Kjetil Berge contributed equally to this article.