A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels

Cancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4‐nitrobenzenesulfonyl (DNS) protected 2‐hydroxyisophthalamide system that exploits GSH for its activation into free 2‐hydroxyisophthalamide forming supramolecular M⁺/Cl^? channels. Better permeation of the DNS protected compound into MCF‐7 cells compared to the free 2‐hydroxyisophthalamide and GSH‐activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH‐activatable transport‐mediated cell death was further validated in rat insulinoma cells (INS‐1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF‐7 cells with efficiency similar to that of the anticancer drug doxorubicin.     

High-throughput virtual screening and preclinical analysis identifies CB-1, a novel potent dual B-Raf/c-Raf inhibitor,effective against wild and mutant variants of B-Raf expression in colorectal carcinoma

Paradoxical Raf activation via Raf dimerization is a major drawback of wild/mutant B-Raf inhibitors. Herein, we report that CB-1 a novel, potent B-Raf/c-Raf dual inhibitor, effective against colon cancer cells, irrespective of their genetic status. High-throughput virtual screening of the ChemBridge library against wild B-Raf (B-Raf^WT), mutant B-Raf (B-Raf^V600E), and c-Raf was performed using an automated protocol with the AutoDock-VINA. Caco-2 and HT-29 cells were used. Of the 23,365 compounds screened computationally, CB-1 showed the highest binding energy towards B-Raf^WT with a ΔG_binding score of ? 13.0 kcal/mol. The compound was also predicted to be effective against B-Raf^V600E and c-Raf molecules with ΔG_binding energies of ? 10.6 and ? 10.1 kcal/mol, respectively. The compound inhibited B-Raf^WT, B-Raf^V600E and c-Raf kinases with IC₅₀ values of 27.13, 51.70, and 40.23 nM, respectively. The GI₅₀ value of CB-1 was 247.9 nM in B-Raf^WT-expressing Caco-2 cells and 352.4 nM in B-RafV600E-expressing HT-29 cells. Dose-dependent increases in total apoptosis and G₁ cell cycle phase arrest was observed in CB-1-treated colon cancer cells. The compound decreased B-Raf expression in both wild and mutant colon cancer cells. CB-1, a novel, potent dual B-Raf/c-Raf inhibitor was effective against colon cancer cells bearing wild-type and mutant variants of B-Raf expression.

     

Seriniquinones as Therapeutic Leads for Treatment of BRAF and NRAS Mutant Melanomas

Isolated from the marine bacteria Serinicoccus sp., seriniquinone (SQ1) has been characterized by its selective activity in melanoma cell lines marked by its modulation of human dermcidin and induction of autophagy and apoptosis. While an active lead, the lack of solubility of SQ1 in both organic and aqueous media has complicated its preclinical evaluation. In response, our team turned its effort to explore analogues with the goal of returning synthetically accessible materials with comparable selectivity and activity. The analogue SQ2 showed improved solubility and reached a 30–40-fold greater selectivity for melanoma cells. Here, we report a detailed comparison of the activity of SQ1 and SQ2 in SK-MEL-28 and SK-MEL-147 cell lines, carrying the top melanoma-associated mutations, BRAF^V600E and NRAS^Q61R, respectively. These studies provide a definitive report on the activity, viability, clonogenicity, dermcidin expression, autophagy, and apoptosis induction following exposure to SQ1 or SQ2. Overall, these studies showed that SQ1 and SQ2 demonstrated comparable activity and modulation of dermcidin expression. These studies are further supported through the evaluation of a panel of basal expression of key-genes related to autophagy and apoptosis, providing further insight into the role of these mutations. To explore this rather as a survival or death mechanism, autophagy inhibition sensibilized BRAF mutants to SQ1 and SQ2, whereas the opposite happened to NRAS mutants. These data suggest that the seriniquinones remain active, independently of the melanoma mutation, and suggest the future combination of their application with inhibitors of autophagy to treat BRAF-mutated tumors.     

High-sensitivity PCR method for detecting BRAF V600Emutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles

The response to epidermal growth factor receptor (EGFR)-targeted therapy in metastatic colorectal cancer (mCRC) is variable because of intra-tumor heterogeneity at the genetic level, and consequently, it is important to develop sensitive and selective assays to predict patient responses to therapy. Low-abundance BRAF V600E mutations are associated with poor response to treatment with EGFR inhibitors. We developed a method for the detection of BRAF V600E mutations in mCRC using real-time wild-type blocking PCR (WTB-PCR), in which a chimera composed of locked nucleic acids and DNA is incorporated to amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. Mixing experiments showed that this method is exquisitely sensitive, with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000. To demonstrate the applicability of this approach for mCRC patients, we assessed the V600E mutations in 50 clinical cases of mCRC by real-time WTB-PCR. The percentage of patients with V600E mutation as determined by WTB-PCR (16 %, 8/50) was higher than by traditional PCR (10 %, 5/50), suggesting an increased sensitivity for WTB-PCR. By calculating the ΔC _q for real-time traditional PCR, which amplifies all BRAF alleles, versus WTB-PCR, which selectively amplifies mutant BRAF, we demonstrated that among the V600E-positive mCRC patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 0.05 to 52.32 %. In conclusion, WTB-PCR provides a rapid, simple, and low-cost method to detect trace amounts of mutated BRAF V600E gene.     

Design,synthesis, docking and mechanistic studies of new thiazolyl/thiazolidinylpyrimidine-2,4-dione antiproliferative agents

In this article, we display on the synthesis and biological evaluation of a new series of thiazolylpyrimidine 3a-l and thiazolidinylpyrimidine derivatives 5a-e. The structures of the new compounds were confirmed by using different spectral techniques including NMR, IR, mass spectroscopy in addition to elemental analyses. The cell viability of the new compounds was assessed against normal human mammary gland epithelial (MCF-10A) cell line. Data revealed that none of the compounds examined exhibited cytotoxic effects, and the cell viability for the compounds examined at 50 µM was greater than 87%. The antiproliferative activity of 3a-l and 5a-e was evaluated against four human cancer cell lines where the compounds showed promising activity. The most potent derivatives were compounds 3a, 3c, 3f, 3i, and 5b with GI₅₀ values ranging from 0.90 µM to 1.70 µM against the four cancer cell lines in comparison to doxorubicin (GI₅₀ = 1.10 µM). Compounds 3a, 3c and 3i showed potent antiproliferative activity with dual inhibitory action against EGFR and BRAF^V600E. Compounds 3a, 3c, and 3i demonstrated promising AutoDock scores towards EGFR and BRAF^V600E with values of ? 9.1 and ? 8.6, ?9.0 and ? 8.5, and ? 8.4 and ? 8.0 kcal/mol, respectively. The physicochemical and pharmacokinetic characteristics of 3a, 3c, and 3i were anticipated, demonstrating their oral bioavailability.     

Role of galectin-3 in tumour metastasis

We used metastatic variant of B16 melanoma (B16F1) to study lung colonization galectin-3-deficient (gal-3^−/−) C57BL/6 mice. In vivo study showed that compared with gal-3^+/+ mice, gal-3^−/−mice exhibited resistance to lung colonization of B16F1 melanoma cells (p < 0.03). In vitro assays showed higher number of attached malignant cells in the tissue section derived from gal-3^+/+ mice (p < 0.001) and tumor specific cytotoxicity of lymphoid cells of tumour inoculated gal-3^−/− suggesting that galectin-3 is considered as therapeutic target.     

Specific Targeting of PEGylated Liposomal Doxorubicin (Doxil®) to Tumour Cells Using a Novel TIMP3 Peptide

Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil^®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil^® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil^® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil^® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers.     

Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen,FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.     

Ethidium Binding to Salmonella enterica ser. Typhimurium Cells and Salmon Sperm DNA

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et⁺) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et⁺ concentration. The results of our experiments indicated that Et⁺ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et⁺, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et⁺. Measurements of Et⁺ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et⁺ considerably decreases in the presence of Mg²⁺, Polymyxin B or when DNA is incubated in high ionic strength media.     

Excess molar volumes of 1-nonanol withn-heptane at 25, 35 and 45°C1

The excess molar volumes V _m ^E at atmospheric pressure and at 25, 35 and 45°C for binary mixtures of 1-nonanol, with n-heptane have been obtained over the whole mole fraction range from densities measured with a vibrating-tube densimeter. The measurements at 25°C were extended to high dilution of 1-nonanol. The V _m ^E are sigmoid for the three temperatures, with a small maximum at low mole fractions of the alkanol. The absolute values of V _m ^E increase with temperature from 25 to 45°C.Communicated in part at the 4^th International Conference on Thermodynamics of Solution of Non-Electrolytes, Santiago de Compostela, Spain (1989).     

Development of a novel class of B-Raf(V600E)-selective inhibitors through virtual screening and hierarchical hit optimization

Oncogenic mutations in critical nodes of cellular signaling pathways have been associated with tumorigenesis and progression. The B-Raf protein kinase, a key hub in the canonical MAPK signaling cascade, is mutated in a broad range of human cancers and especially in malignant melanoma. The most prevalent B-Raf(V600E) mutant exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, we describe the development of novel B-Raf(V600E) selective inhibitors via multi-step virtual screening and hierarchical hit optimization. Nine hit compounds with low micromolar IC(50) values were identified as B-Raf(V600E) inhibitors through virtual screening. Subsequent scaffold-based analogue searching and medicinal chemistry efforts significantly improved both the inhibitor potency and oncogene selectivity. In particular, compounds and possess nanomolar IC(50) values with selectivity for B-Raf(V600E)in vitro and exclusive cytotoxicity against B-Raf(V600E) harboring cancer cells.     

Nocardioazines: a novel bridged diketopiperazine scaffold from a marine-derived bacterium inhibits P-glycoprotein

An Australian marine sediment-derived isolate, Nocardiopsis sp. (CMB-M0232), yielded a new class of prenylated diketopiperazine, indicative of the action of a uniquely regioselective diketopiperazine indole prenyltransferase. The bridged scaffold of nocardioazine A proved to be a noncytotoxic inhibitor of the membrane protein efflux pump P-glycoprotein, reversing doxorubicin resistance in a multidrug resistant colon cancer cell.     

Melatonin prevents doxorubicin-induced cardiotoxicity through suppression of AMPKα2-dependent mitochondrial damage

The clinical application of doxorubicin, one of the most effective anticancer drugs, has been limited due to its adverse effects, including cardiotoxicity. One of the hallmarks of doxorubicin-induced cytotoxicity is mitochondrial dysfunction. Despite intensive research over recent decades, there are no effective approaches for alleviating doxorubicin-induced cytotoxicity. Melatonin, a natural hormone that is primarily secreted by the pineal gland, is emerging as a promising adjuvant that protects against doxorubicin-induced cytotoxicity owing to its pharmaceutical effect of preserving mitochondrial integrity. However, the underlying mechanisms are far from completely understood. Here, we provide novel evidence that treatment of H9c2 cardiomyoblasts with doxorubicin strongly induced AMP-activated protein kinase α2 (AMPKα2), which translocated to mitochondria and interfered with their function and integrity, ultimately leading to cellular apoptosis. These phenomena were significantly blocked by melatonin treatment. The levels of AMPKα2 in murine hearts were tightly associated with cardiotoxicity in the context of doxorubicin and melatonin treatment. Therefore, our study suggests that the maintenance of mitochondrial integrity is a key factor in reducing doxorubicin-induced cytotoxicity and indicates that AMPKα2 may serve as a novel target in the design of cytoprotective combination therapies that include doxorubicin.Subject terms: Biochemistry, Diseases     

Doxorubicin Encapsulated in TPGS-Modified 2D-Nanodisks Overcomes Multidrug Resistance

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d -α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.     

Application of the Prigogine-Flory-Patterson theory part II. Mixtures of a bicyclic compound,benzene, cyclohexane,n-hexane with a 1-alkene and a 1-alkyne

The Prigogine-Flory-Patterson theory of liquid mixtures has been applied to the H _m ^E and V _m ^E for binary mixtures of an n-alkane with decalin, bicyclohexyl, tetralin, cyclohexylbenzene, benzene, cyclohexane and n-hexane with 1-hexene, 1-hexyne, 1-heptene and 1-heptyne. Furthermore the Prigogine-Flory theory has been used to predict activity coefficients at infinite dilution from the experimentally determined H _m ^E at 25°C for the mixtures 1-hexene, 1-hexyne, 1-heptene, 1-heptyne with decalin, bicyclohexyl, tetralin and cyclohexylbenzene. The predictions are compared to experimental results.     

Serjanic Acid Glycosides from Chenopodium hybridum L. with Good Cytotoxicity and Selectivity Profile against Several Panels of Human Cancer Cell Lines

Two triterpene saponins, including a novel serjanic acid derivative, were isolated from Chenopodium hybridum L. (Amaranthaceae) aerial parts. Their structures were elucidated by a combination of spectroscopic methods (MS, 1D and 2D NMR). Both compounds were evaluated for cytotoxicity and selectivity on skin, prostate, gastrointestinal, thyroid and lung cancer cells. Their effect was dose and time-dependent with varied potency, the highest against prostate PC3 and melanoma WM793, where IC₅₀ was lower than the reference drug doxorubicin. Structure–activity relationship is briefly discussed.     

Drug Self‐Delivery Systems Based on Hyperbranched Polyprodrugs towards Tumor Therapy

Amphiphilic hyperbranched polyprodrugs (DOX‐S‐S‐PEG) with drug repeat units in hydrophobic core linked by disulfide bonds were developed as drug self‐delivery systems for cancer therapy. The hydroxyl groups and the amine group in doxorubicin (DOX) were linked by 3,3′‐dithiodipropanoic acid as hydrophobic hyperbranched cores, then amino‐terminated polyethylene glycol monomethyl ether (mPEG‐NH₂) as hydrophilic shell was linked to hydrophobic cores to form amphiphilic and glutathione (GSH)‐responsive micelle of hyperbranched polyprodrugs. The amphiphilic micelles can be disrupted under GSH (1 mg mL^?1) circumstance. Cell viability of A549 cells and 293T cells was evaluated by CCK‐8 and Muse Annexin V & Dead Cell Kit. The disrupted polyprodrugs maintained drug activity for killing tumor cells. Meanwhile, the undisrupted polyprodrugs possessed low cytotoxicity to normal cells. The cell uptake experiments showed that the micelles of DOX‐S‐S‐PEG were taken up by A549 cells and distributed to cell nuclei. Thus, the drug self‐delivery systems with drug repeat units in hydrophobic cores linked by disulfide bonds showed significant special advantages: 1) facile one‐pot synthesis; 2) completely without toxic or non‐degradable polymers; 3) DOX itself functions as fluorescent labeled molecule and self‐delivery carrier; 4) drug with inactive form in hyperbranched cores and low cytotoxicity to normal cells. These advantages make them excellent drug self‐delivery systems for potential high efficient cancer therapy.     

The effect of steel modifying with zinc oxyethylidenediphosphonate on its passivation in solutions of certain inhibitors

It is shown that a solution of zinc complex of 1-hydroxyethane-1,1-diphosphonic acid can be used for modifying the surface of low-carbon steel in order to enhance its passivation by aqueous solutions of other inhibitors (1,2,3-benzotriazole, sodium alkylcarboxylate, and their mixture). The results of accelerated tests of protectability of nanosized passivating coatings are compared. To assess the efficiency of steel protection by electrochemical tests, the difference in pit formation potentials ΔE = E _pt ⁱⁿ ? E _pt ^sup are determined from polarization curves in borate buffer solutions containing NaCl in the absence (E _pt ^sup ) and in the presence (E _pt ⁱⁿ ) of the inhibitor. The direct corrosion tests are carried out in cells and in the G-4 humidity chamber (GOST (State Standard) 9.054–75) where the time of the appearance of first indications of corrosion on samples τ_cor serves as the criterion of efficient protection. Three groups of coatings can be distinguished: with weak protectability characterized by ΔE < 0.20 V and τ_cor < 6.25 days; with medium protectability and ΔE = 0.20–0.40 V with τ_cor reaching 9 days; and the with high protecting efficiency and ΔE > 0.40 V where τ_cor can be even longer than 25 days.     

Darstellung und spektroskopische Charakterisierung von Di(halogeno)phthalocyaninato(1–)rhodium(III), [RhX2Pc1−] (X = Cl,Br, I)

Synthesis and Spectroscopical Characterization of Di(halo)phthalocyaninato(1–)rhodium(III), [RhX₂Pc^1?] (X = Cl, Br, I) Bronze-coloured di(halo)phthalocyaninato(1–)-rhodium(III), [RhX₂Pc^1?] (X = Cl, Br) and [RhI₂Pc^1?] · I₂ is prepared by oxidation of (ⁿBu₄N)[RhX₂Pc^2?] with the corresponding halogene. Irrespective of the halo ligands, two irreversible electrode reactions due to the first ringreduction (E_R = ?0,90 V) and ringoxidation (E_O = 0,82 V) are present in the cyclovoltammogram of (ⁿBu₄N)[RhX₂Pc^2?]. The optical spectra show typical absorptions of the Pc^1?-ligand at 14.0 kK and 19.1 kK. Characteristic vibrational bands are at 1 366/1 449 cm^?1 (i. r.) and 569/1 132/1 180/1 600 cm^?1 (resonance Raman (r. r.)). The antisym. (Rh? X)-stretching vibration is observed at 294 cm^?1 (X = Cl), 240 cm^?4 (Br) and 200 cm^?1 (I). Only the sym. (Rh? I)-stretching vibration at 133 cm^?1 is r. r. enhanced together with a strong line at 170 cm^?1, which is assigned to the (I? I)-stretching vibration of the incorporated iodine molecule. Both modes show overtones and combinationbands.     

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.