Cytotoxic Products Research Articles

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34+ cells

Background aimsNatural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34+ cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. MethodsWe evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. ResultsWe found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. ConclusionsWe are therefore able to generate large numbers of functional NK cells from CB CD34+ cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product.

The effects of various dietary vegetable oil sources on villi morphology and liver aldehydes in young layers

Twenty-six-week-old laying hens (90 Lohmann Classic Brown) received dietary treatments containing canola and linseed oil at 2% inclusion levels and a control diet for 21 days. The 2% dietary linseed oil, which had higher total polyunsaturated fatty acids (ΣPUFA) and linolenic fatty acid contents, resulted in higher liver malondialdehyde and wider villi than the control group. The 2% canola and linseed oil treatments recorded smaller reproductive organs relative to live weight than the control group. It was concluded that young layers might have an anatomical response to high levels of dietary PUFA, which might improve nutrient absorption and cause a biochemical response, resulting in potentially negative cytotoxic aldehydic lipid peroxidation products. Keywords: Intestine morphology, malondialdehyde, n-3, poultry, PUFA

From the discovery of microbial Mep-Amt ammonium transporters to human Rhesus factors

Ammonium, ubiquitous on Earth, plays major and distinct roles in most organisms. While it can be a nitrogen source for many microorganisms and plants, it is a cytotoxic metabolic product actively detoxified by the liver in animals. Furthermore, in the latter, ammonium synthesis in the kidney is involved in acid/base homeostasis. Ammonium transport is ensured by a family of proteins, called Mep-Amt-Rh. This family is conserved in all domains of life and comprises the human Rh factors, notably known in transfusional medicine. While the study of bacterial, fungal and vegetal Mep-Amt transporters reveals a fine-tuned and rapid regulation of these proteins in function of environmental changes, the regulation of animal Rh proteins has been poorly addressed. This review notably highlights the importance of the yeast model in the study of the regulation of these proteins as well as in the functional characterization of Mep-Amt-Rh members of diverse origins.

Cost avoidance from dose rounding biologic and cytotoxic antineoplastics.

Background To reduce product wastage, our institution allows automatic dose rounding of biologic and cytotoxic anticancer agents. The purpose of this project was to determine the actual annual cost avoidance due to pharmacist-managed automatic dose rounding of anticancer treatments. Methods Financial impact was assessed within the context of our departmental standard work which supports automatic dose rounding of biologic anticancer agents (±10%) and cytotoxic anticancer agents (±5%) to the nearest vial size for body surface area- or weight-based doses. Exclusions to automatic dose rounding include multiple dose vial products, pediatric orders, clinical trial drugs, and parenteral busulfan. The amount of cost avoidance for each rounded dose was determined using the product acquisition cost of the smallest available product amount. Data were collected from anticancer treatment orders for the fiscal year 1 July 2013 to 30 June 2014. Results A total of 6216 doses of anticancer drugs were checked for dose rounding during the period of data collection. Almost $200,000 in product acquisition cost was avoided with pharmacist-managed automatic dose rounding. Six different biologic products accounted for approximately 7% of the total doses analyzed and 78% of the cost avoidance. Fifteen drugs comprised the array of cytotoxic agents rounded. Approximately, 37% and 4% of the biologic and cytotoxic doses were rounded up to the vial size. Conclusion Routine dose rounding of biologic anticancer agents (±10%) and cytotoxic products (±5%) achieved cost avoidance through reduction of drug wastage at our institution.

Catalytic SN2'- and Enantioselective Allylic Substitution with a Diborylmethane Reagent and Application in Synthesis.

A catalytic method for the site- and enantioselective addition of commercially available di-B(pin)-methane to allylic phosphates is introduced (pin=pinacolato). Transformations may be facilitated by an NHC-Cu complex (NHC=N-heterocyclic carbene) and products obtained in 63-95 % yield, 88:12 to >98:2 S(N)2'/S(N)2 selectivity, and 85:15-99:1 enantiomeric ratio. The utility of the approach, entailing the involvement of different catalytic cross-coupling processes, is highlighted by its application to the formal synthesis of the cytotoxic natural product rhopaloic acid A.

Catalytic SN2′‐ and Enantioselective Allylic Substitution with a Diborylmethane Reagent and Application in Synthesis

AbstractA catalytic method for the site‐ and enantioselective addition of commercially available di‐B(pin)‐methane to allylic phosphates is introduced (pin=pinacolato). Transformations may be facilitated by an NHC–Cu complex (NHC=N‐heterocyclic carbene) and products obtained in 63–95 % yield, 88:12 to >98:2 SN2′/SN2 selectivity, and 85:15–99:1 enantiomeric ratio. The utility of the approach, entailing the involvement of different catalytic cross‐coupling processes, is highlighted by its application to the formal synthesis of the cytotoxic natural product rhopaloic acid A.

Iterative design of emetine-based prodrug targeting fibroblast activation protein (FAP) and dipeptidyl peptidase IV DPPIV using a tandem enzymatic activation strategy.

There is an urgent need to develop new agents for treating metastatic prostate cancer to overcome multiple drug resistance to the current standard targeted cancer therapy. Emetine is a highly cytotoxic natural product protein synthesis inhibitor, which is toxic to all cell types. Its cytotoxicity can be blocked by derivatizing its N-2' position. Thus emetine can be selectively delivered to cancer cells in the region of metastatic cancer as a prodrug that will be activated by an enzyme selectively overexpressed within the metastatic tumor microenvironment. In this work, we convert emetine to a prodrug activatable by the fibroblast activation protein (FAP), a serine protease overexpressed by the carcinoma associated fibroblasts. By using an iterative structure-activity relationship strategy, several peptidyl emetine prodrug analogs (1-11) were synthesized by chemical derivatization of emetine at its N-2' position and tested for in-vitro activation by FAP. The lead prodrug 11 is made up of a DPPIV activatable prodrug precursor 10 (Ala-Pro-PABC-Emetine) coupled to FAP substrate (Ala-Ser-Gly-Pro-Ala-Gly-Pro). Activation assays of the prodrugs were performed in purified FAP, DPPIV, FBS, and human serum and were analyzed by LCMS. In vitro cytotoxicity assays of these prodrugs are carried out in prostate (LNCaP, PC3) and breast (MCF7 and MDA-MB-231) cancer cell lines. The prodrugs are also tested in normal immortalized human prostatic epithelial cell line (PrEC). The lead FAP activated emetine prodrug 11 is activated to emetine in tandem by FAP and DPPIV in about 70% conversion within 24 hr. In prostate and breast cancer cell lines treated with prodrug 11, it is found to be equipotent with emetine in the presence of FAP and DPPIV. However, in the PrEC cell line grown in serum free media, prodrug 11 is more than 200-fold less cytotoxic than emetine in the absence of FAP and DPPIV. This FAP activated prodrug of cytotoxic agent emetine further shows the crucial role of the N-2' position of emetine in controlling its cytotoxicity. Significantly reduced toxicity observed in the PrEC cell line in the absence of FAP and DPPIV shows that prodrug 11 could be systemically delivered to regions of metastatic prostate cancer or other solid tumor for activation by cancer selective enzymes within the cancer microenvironment, such as FAP that is overexpressed by the carcinoma-associated fibroblasts. The two-step tandem enzymatic activation of prodrug 11 by FAP and DPPIV is a strategy for overcoming steric hindrance.

Biliverdin protects against cisplatin-induced apoptosis of renal tubular epithelial cells.

Biliverdin (BV) has long been thought to be a cytotoxic metabolic waste product. It has also been demonstrated to have important cytoprotective functions during oxidative stress. The present study aimed to examine the cytoprotective effect of BV on NRK-52E cells, a proximal tubular cell line derived from rat kidney. Cells were treated with 50 µmol/L cisplatin for 24 h (cisplatin group) or pre-treated with BV for 30 min, then with 50 µmol/L cisplatin for 24 h (cisplatin+BV group). Those given no treatment served as a control. Cell apoptosis was evaluated by flow cytometry and cell viability by Cell Counting Kit-8 (CCK-8). The protein expressions of cleaved caspase3, Bax and Bcl-2 were assessed by Western blotting. Reactive oxygen species (ROS) levels were measured using carboxydichlorodihydrofluorescein diacetate (H2DCF). The results showed that cisplatin induced the apoptosis of NRK-52E cells, decreased cell viability, and increased the formation of ROS by upregulating the expression of cleaved caspase3 and Bax and decreasing Bcl-2 protein expression. These effects could be significantly reversed by pretreatment with BV. It was concluded that BV can protect against cisplatin-induced cell apoptosis through the anti-oxidative effects.

Synthesis of the C1–C13 Fragment of Mandelalide A

This communication describes the synthesis of the fully functionalized C1 to C13 segment (southern part) of highly potent cytotoxic marine natural product mandelalide A. Pd-catalyzed oxa-­Michael cyclization followed by stereoselective reduction of the C7-ketone were carried out to construct the tetrahydropyran unit with required stereochemistry. The α,β-unsaturated ester moiety was constructed via cross-metathesis and the vinyl iodide was installed through modified Takai olefination.

Synthetic studies of callyspongiolide: synthesis of the macrolactone core of the molecule

A concise synthetic strategy has been developed for the synthesis of the macrolactone core 2 of a unique polyketide callyspongiolide 1. The key features of the strategy included an Evan's asymmetric alkylation, diastereoselective Michael type alkylation, Brown's asymmetric allylation reaction, an allylic alkylation of an activated Z-allylic alcohol and an intramolecular Z-selective intramolecular H-W-E olefination.

Diastereoseletive Transannular Oxa-Conjugate Addition Generates the 2,6-cis-Disubstituted Tetrahydropyran of Neopeltolide.

Transannular 2,6-disubstituted pyrans, like the one found in the cytotoxic marine natural product neopeltolide, are a key functional group in many polyketides. While oxa-conjugate additions have been shown to provide direct and rapid access to tetrahydropyrans in acyclic neopeltolide intermediates, a transannular strategy for construction of this ring system in a macrocyclic core has not been investigated. In this study, we demonstrate that a transannular oxa-conjugate addition strategy is a viable approach to the construction of the bicyclic core of neopeltolide. We show that transannular addition occurs readily with an α,β-unsaturated ketone as the Michael acceptor and does not occur when an α,β-unsaturated ester is the Michael acceptor. Our data indicates that oxa-conjugate addition is reversible and that the stereochemical outcome can be under thermodynamic control. Using computational chemistry, we show that the lowest energy diastereomer is the desired cis-pyran found in neopeltolide, and we experimentally demonstrate that the trans and cis diastereomers are interconvertible under reaction conditions with the cis-pyran product predominating. This oxa-conjugate addition strategy should provide a viable route to accessing the fully elaborated macrocyclic core of neopeltolide.

Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.

Abstract B170: Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models

Abstract Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecule cytotoxic drugs and have the potential to provide significant efficacy as a treatment for cancer. The objective of this work was to identify potent new cytotoxic ADC payloads that can be used to target diverse tumor types. Here we report for the first time the discovery of fully synthetic tubulysin payloads which belong to a class of highly cytotoxic natural products that disrupt the cellular microtubule network leading to apoptosis of tumor cells. Our fully synthetic tubulysin payloads are comprised of: (i) a tubulysin warhead that displays pM potency, (ii) a protease cleavable amino-acid sequence and (iii) a tether bearing a reactive maleimide group. Tubulysin-based ADCs were generated via site-specific conjugation of these payloads to cysteines engineered into antibodies against cancer antigen target oncofetal protein 5T4. The resulting ADCs showed potent in vitro cell killing and in vivo efficacy in multiple solid tumor xenograft models including prostate cancer, non-small cell lung adenocarcinoma, breast cancer and gastric carcinoma. Furthermore, specific structural features of the tubulysin warhead, linker design and antibody engineering were shown to impact the overall in vitro and in vivo properties of the ADCs. Thus, these synthetic tubulysin payloads represent novel microtubule network disrupting compounds that display potent preclinical anti-tumor activity as an ADC that could be advanced to the clinic. Citation Format: Dorin Toader, Jay Harper, Chris Lloyd, Rose Marwood, David Bannister, Shenlan Mao, Cui (Tracy) Chen, Haihon (Helen) Zhong, Vahe Bedian, Fengjiang Wang, Lakshmaiah Gingipalli, Melisa Vasbinder, Pamela Thompson, Ryan Fleming, Byniam Bezabeh, Nazzareno Dimasi, Changshou Gao, Adeela Kamal. Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B170.

Assessment of Physicochemical Parameters and Storage Stability of Reused Oils

Oils are obtained from nuts and seeds of plants such as corn, groundnut, soyabean, olive, sunflower, cotton seed, palm, coconut and rape seed. Oils are extracted from the nuts or seeds by crushing and by the use of solvent extraction [1]. Deep fat frying is a popular food preparation method, where thermal stressing of polyunsaturated fatty acid, generates high levels of cytotoxic aldehydic products, arising from the fragmentation of conjugated hydroperoxydiene precursors. Such aldehydic compounds are damaging to human health [2]. Cooking oil is chemically decomposed in the cooking process and this decomposition leads to a deterioration in cooking quality, if the oil is kept in use for a long time [3]. Hence the present study was undertaken to assess the physicochemical parameters and storage stability of reused groundnut and palm oils. Results showed that physiochemical characteristics of oil were altered during repeated frying and storage and the changes in sensory attributes were also observed.

Clinical Approach of High Technology Techniques for Control and Elimination of Endodontic Microbiota.

The main goal in endodontic treatment is to eradicate or at least reduce intraradicular microbial population to levels that are more compatible with periapical lesions healing process. Since endodontic infections are polymicrobial in nature, intraradicular survival of endodontic microbiota and their pathogenic properties are influenced by a combination of their virulence factors. The purpose of this article is to review the endodontic microbiota and their respective virulence attributes, as well as perform a literature review of the effects of disinfection procedures in the treatment of endodontic infections to gain best practices. Conventional technique for root canal preparation includes mechanical debridement and application of antimicrobial irrigants. Recently, laser irradiation has been used to enhance the results of root canal treatment through its thermal effect. To reduce thermal side effects, laser activated irrigation (LAI) and photon induced photoacoustic streaming (PIPS) were introduced. Antimicrobial photodynamic therapy (aPDT) by photochemical reaction uses light at a specific wavelength to activate a nontoxic photosensitizer (PS) in the presence of oxygen to produce cytotoxic products. Different PSs are used in dentistry including methylene blue (MB), toluidine blue O (TBO), indocyanine green (ICG) and curcumin. Among different options, ICG could be the best choice due to its peak absorption at wavelength of 808 nm, which coincides with the commercial diode laser devices. Also, this wavelength has more penetration depth compared to other wavelengths used in aPDT.

P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvement of peroxynitrite.

BackgroundThe NLR family, pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in intracerebral hemorrhage (ICH)-induced inflammatory injury, and the purinergic 2X7 receptor (P2X7R) is upstream of NLRP3 activation. This study aimed to investigate how P2X7R functions in ICH-induced inflammatory injury and how the receptor interacts with the NLRP3 inflammasome.MethodsRats were treated with P2X7R small interfering RNA (siRNA) 24 h before undergoing collagenase-induced ICH. A selective P2X7R inhibitor (blue brilliant G, BBG) or a peroxynitrite (ONOO−) decomposition catalyst (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) [FeTPPS]) was injected 30 min after ICH. Brain water content, hemorrhagic lesion volume, and neurological deficits were evaluated, and western blot, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were carried out.ResultsStriatal P2X7R and NLRP3 inflammasomes were activated after ICH. Gene silencing of P2X7R suppressed NLRP3 inflammasome activation and interleukin (IL)-1β/IL-18 release and significantly ameliorated brain edema and neurological deficits. Additionally, enhanced NADPH oxidase 2 (NOX2, gp91phox) and inducible nitric oxide synthase (iNOS), as well as their cytotoxic product (ONOO−) were markedly attenuated by BBG treatment following ICH. This was accompanied by downregulations of the inflammasome components, IL-1β/IL-18 and myeloperoxidase (MPO, a neutrophil marker). Most importantly, inflammasome activation and IL-1β/IL-18 release were significantly inhibited by ONOO− decomposition with FeTPPS.ConclusionsOur findings implicate that P2X7R exacerbated inflammatory progression and brain damage in ICH rats possibly via NLRP3 inflammasome-dependent IL-1β/IL-18 release and neutrophil infiltration. ONOO−, a potential downstream signaling molecule of P2X7R, may play a critical role in triggering NLRP3 inflammasome activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0409-2) contains supplementary material, which is available to authorized users.

Mitochondrial respiratory dysfunction due to the conversion of substituted cathinones to methylbenzamides in SH-SY5Y cells.

The increased use of cathinone-type designer drugs, known as legal highs, has led to concerns about their potential neurotoxicity due to their similarity to methamphetamine (METH). Therefore, closer investigations of their toxic effects are needed. We investigated the effects of the cathinones 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxymethcathinone (MDMC) and the amphetamine METH on cytotoxicity and mitochondrial respiration in SH-SY5Y neuroblastoma cells. We also investigated the contribution of reactive species, dopamine, Bcl-2 and tumor necrosis factor α (TNFα) on toxicity. Finally, we investigated the effect of cathinone breakdown products using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry and studied their involvement in toxicity. We observed dose-dependent increases in cytotoxicity and decreases in mitochondrial respiration following treatment with all cathinones and amphetamines. Glutathione depletion increases amphetamine, but not cathinone toxicity. Bcl-2 and TNFα pathways are involved in toxicity but dopamine levels are not. We also show that cathinones, but not amphetamines, spontaneously produce reactive species and cytotoxic methylbenzamide breakdown products when in aqueous solution. These results provide an important first insight into the mechanisms of cathinone cytotoxicity and pave the way for further studies on cathinone toxicity in vivo.

The effect of plasma surface modification on the biodegradation rate and biocompatibility of a poly(butylene succinate)-based copolymer

In this contribution, a poly(butylene succinate)-based copolymer containing thioether linkages (P(BS85BTDG15)) has been synthesized and characterized from the molecular, thermal and mechanical point of view. The results have been compared to the parent homopolymer. Films obtained both by PBS and P(BS85BTDG15) have been subjected to surface modification by means of non-thermal plasma surface treatment and the effect on physic/mechanical properties has been considered. Hydrolytic degradation rate under physiological conditions and in vitro biocompatibility of treated and non-treated polymeric films have been also investigated.The introduction of thioether linkages and the plasma etching enhanced the polymer surface wettability, thus resulting in an increased hydrolytic degradation rate. On the other hand, bulk properties were not significantly affected.Biocompatibility assays highlighted the absence of potentially cytotoxic products into the culture medium and proved that the investigated polymeric films can support cell adhesion and proliferation.

Structural basis of Escherichia coli nitroreductase NfsB triple mutants engineered for improved activity and regioselectivity toward the prodrug CB1954

Escherichia coli nitroreductase NfsB coupled with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] has great anti-cancer potential. However, its efficacy is limited by the low catalytic efficiency of NfsB against CB1954. In this paper, we show that substitution with a tryptophan at residue 124 strongly increases the selectivity toward the 4-NO2 group of CB1954, generating the more cytotoxic 4-hydroxylamine product. To further improve the activity, the F124W mutation and three previously reported beneficial mutations were combined randomly into double and triple mutants. Steady-state kinetic studies showed that they all exhibited enhanced activity toward CB1954. Two triple mutants, T41L/N71S/F124W and F123A/N71S/F124W, show a 9.2–17.2-fold increase in kcat/Km compared with the wild-type and selectively reduce the 4-NO2 group of CB1954. The crystal structure of F123A/N71S/F124W was resolved. Comparison with the reported NfsB structures revealed that the F124W mutation may provide a stronger hydrophobic interaction with the aziridinyl group of CB1954, which was in favor of the reduction of the 4-NO2 group; while the F123A and T41L mutations increased the kcat values in two different ways, leading to improved enzyme activity.

Commiphora gileadensis sap extract induces cell cycle-dependent death in immortalized keratinocytes and human dermoid carcinoma cells

Commiphora gileadensis is an aromatic plant traditionally used in the Middle East as a common remedy for pain and inflammation. Recently, several studies have reported on its anti-proliferative effect in cancer cell lines. Yet, scientific evidence regarding the other properties of C. gileadensis is limited. The aim of the current study was to investigate the cytotoxic action of C. gileadensis sap extracts (CgSE) on human epidermal cells and skin tissues and to ascertain its mode of action. The effects of the extract on cell viability and apoptosis were evaluated on immortalized keratinocytes, human dermal fibroblasts, human dermoid carcinoma cells and on human ex vivo skin cultures. CgSE significantly reduced the viability of both immortalized and transformed epidermal cells by 64% and 68%, respectively. However, normal fibroblasts and human skin organ cultures were protected from this effect. The cytotoxicity was coupled with the induction of capsase-3 and the morphological characteristics of apoptosis. In addition, CgSE-induced apoptosis did not occur in cells in the pre-replicative, G1 phase, but only at later phases involved in DNA replication and cell division. HPLC analysis of CgSE showed only negligible traces of β-caryophyllene, a secondary metabolite previously isolated from C. gileadensis and reported to be cytotoxic. Therefore, additional active compound(s) may be involved in the anti-proliferative phenomenon of the extract. The authors concluded that CgSE contains cytotoxic products which specifically target proliferating cells in a cell cycle-dependent manner, and hence may be relevant for the treatment of skin disorders characterized by hyperproliferation, such as skin cancer and psoriasis.