DNA Flow Cytometry Analysis Research Articles

One-Pot Synthesis of New Bipyridine and Yerpyridine Derivatives: Anti-proliferative Evaluation, DNA Flow Cytometry Analysis, and Molecular Docking Study

Some new bipyridine derivatives were synthesized using 2-oxo-4-(pyridin-3-yl)-6-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile 1a. While terpyridine derivatives were synthesized using 2-oxo-4-(pyridin-3-yl)-6-(pyridin-4-yl)-1,2-dihydropyridine-3-carbonitrile 1b as starting material. Alkylation of compounds 1a, b gave the alkyl derivatives 2a, b which converted into aceto hydrazides 3a,b. Treating the aceto hydrazides with ethyl cyanoacetate afforded 2'-(2-(3,5-dioxopyrazolidin-1-yl)-2-oxoethoxy)-6'-(thiophen-2-yl)-3,4'-bipyridine-3'-carbonitrile 4a and 6'-[2-(3,5-dioxopyrazolidin-1-yl)-2-oxoethoxy]-(3,4':2',4''-terpyridine)-5'-carbonitrile 4b. While treatment of 3a, b with ethyl benzoylacetate afforded the carboniltrile derivatives 2'-(2-oxo-2-(5-oxo-3-phenyl-4,5-dihydropyrazol-1-yl)ethoxy)-6'-(thiophen-2-yl)-3,4'-bipyridine-3'-carbonitrile 5a and 6'-[2-oxo-2-(5-oxo-3-phenyl-4,5-dihydropyrazol-1-yl)ethoxy]-(3,4':2',4''-terpyridine)-5' carbonitrile 5b. Treating compounds 3a, and b with two aromatic aldehydes (3,4-dihydroxy benzaldehyde, 4-chlorobenzaldehyde) afforded Schiff bases 6a-d. Finally, treatment of 3a, b with p-toluenesulfonyl chloride (Tosyl chloride) afforded the corresponding benzene sulfonohydrazide 7a, b. IR, 1HNMR, 13CNMR, MS, and elemental analysis confirmed the synthesized compounds. The anti-cancer activity of the compounds was investigated against the breast cancer (MCF-7) cell line. Compound 6b demonstrated the strongest cytotoxic activity among the tested compounds and it is more active than the reference drug 5-fluorouracil (5-FU), so that, it is subjected to further investigations, where its effect on the cell cycle distribution in the MCF-7 cell line, its ability to inhibit Topoisomerase II, the level of Caspase‐3/7 green flow cytometry assay was investigated. Also, Molecular operating environmental docking (MOE) has been studied and found that the inhibition efficiency could be organized as follows (S, Kcal/mol) values: 5b ˃ 6d ˃ 3a ˃ 3b ˃ 6b, while compound 5a did not show any interaction data belonging to the shaped complex with 1cca. Hence, most synthesized compounds can be used as anti-cancer and anti-oxidant agents.

Flow Cytometric Ploidy Analysis in Acute Lymphoblastic Leukemia and Plasma Cell Myeloma

AbstractIdentification of underlying cytogenetic (CG) aberrancies plays a significant role in risk stratification of hematological malignancies. These abnormalities can be due to aberrancies that affect the number or structure of chromosomes. Numerical chromosomal abnormalities are called aneuploidies, which result from either gain or loss of whole chromosomes. Ploidy assessment by CG is a laborious and less sensitive technique. With the aid of fluorescent nucleic acid binding dyes, the total DNA content and different phases of the cell cycle specific to any population of interest can be deciphered and analyzed by flow cytometry (FCM). DNA index (DI), a parameter derived by FCM DNA analysis, is equivalent to conventional CG-based ploidy assessment. In this study, the technical aspects and implications of FCM DNA assessment among patients diagnosed with acute lymphoblastic leukemia and plasma cell myeloma are discussed.

Design, Synthesis, Anticancer Evaluation and Molecular Modeling Studies of New Thiazolidinone-Benzoate Scaffold as EGFR Inhibitors, Cell Cycle Interruption and Apoptosis Inducers in HepG2.

Synthesis of new anticancer candidates with protein kinases inhibitory potency is a major goal of pharmaceutical science and synthetic research. This current work represents the synthesis of a series of substituted benzoate-thiazolidinones. Most prepared thiazolidinones were evaluated in vitro for their potential anticancer activity against three cell lines by MTT assay, and they found to be more effective against cancer cell lines with no harm toward normal cells. Thiazolidinones 5 c and 5 h were further evaluated to be kinase inhibitors against EGFR showing effective inhibitory impact (with IC50 value; 0.2±0.009 and 0.098±0.004 μM, for 5 c and 5 h, respectively). Furthermore, 5 c and 5 h have effects on cell cycle and apoptosis induction capability in HepG2 cell lines by DNA-flow cytometry analysis and annexin V-FITC apoptosis assay, respectively. The results showed that they have effect of disrupting the cell cycle and causing cell mortality by apoptosis in the treated cells. Moreover, molecular docking studies showed better binding patterns for 5 c and 5 h with the active site of the epidermal growth factor receptor (EGFR) protein kinase (PDB code 1M17). Finally, toxicity risk and physicochemical characterization by Osiris method was performed on most of the compounds, revealing excellent properties as possible drugs.

Discovery of new imidazotetrazinones with potential to overcome tumor resistance

We describe the design, organic synthesis, and characterization, including X-ray crystallography, of a series of novel analogues of the clinically used antitumor agent temozolomide, together with their in vitro biological evaluation. The work has resulted in the discovery of a new series of anticancer imidazotetrazines that offer the potential to overcome the resistance mounted by tumors against temozolomide. The rationally designed compounds that incorporate a propargyl alkylating moiety and a thiazole ring as isosteric replacement for a carboxamide, are readily synthesized (gram-scale), exhibit defined solid-state structures, and enhanced growth-inhibitory activity against human tumor cell lines, including MGMT-expressing and MMR-deficient lines, molecular features that confer tumor resistance. The cell proliferation data were confirmed by clonogenic cell survival assays, and DNA flow cytometry analysis was undertaken to determine the effects of new analogues on cell cycle progression. Detailed 1H NMR spectroscopic studies showed that the new agents are stable in solution, and confirmed their mechanism of action. The propargyl and thiazole substituents significantly improve potency and physicochemical, drug metabolism and permeability properties, suggesting that the thiazole 13 should be prioritized for further preclinical evaluation.

Network pharmacology and molecular docking study for biological pathway detection of cytotoxicity of the yellow jasmine flowers

BackgroundThe yellow jasmine flower (Jasminum humile L.) is a fragrant plant belonging to the Oleaceae family with promising phytoconstituents and interesting medicinal uses. The purpose of this study was to characterize the plant metabolome to identify the potential bioactive agents with cytotoxic effects and the underlying mechanism of cytotoxic activity.MethodsFirst, HPLC–PDA-MS/MS was used to identify the potential bioactive compounds in the flowers. Furthermore, we assessed the cytotoxic activity of the flower extract against breast cancer (MCF-7) cell line using MTT assay followed by the cell cycle, DNA-flow cytometry, and Annexin V-FITC analyses alongside the effect on reactive oxygen species (ROS). Finally, Network pharmacology followed by a molecular docking study was performed to predict the pathways involved in anti-breast cancer activity.ResultsHPLC–PDA-MS/MS tentatively identified 33 compounds, mainly secoiridoids. J. humile extract showed a cytotoxic effect on MCF-7 breast cancer cell line with IC50 value of 9.3 ± 1.2 µg/mL. Studying the apoptotic effect of J. humile extract revealed that it disrupts G2/M phase in the cell cycle, increases the percentage of early and late apoptosis in Annexin V-FTIC, and affects the oxidative stress markers (CAT, SOD, and GSH-R). Network analysis revealed that out of 33 compounds, 24 displayed interaction with 52 human target genes. Relationship between compounds, target genes, and pathways revealed that J. humile exerts its effect on breast cancer by altering, Estrogen signaling pathway, HER2, and EGFR overexpression. To further verify the results of network pharmacology, molecular docking was performed with the five key compounds and the topmost target, EGFR. The results of molecular docking were consistent with those of network pharmacology.ConclusionOur findings suggest that J. humile suppresses breast cancer proliferation and induces cell cycle arrest and apoptosis partly by EGFR signaling pathway, highlighting J. humile as a potential therapeutic candidate against breast cancer.

Distribution, Population Systems, and Peculiarities of Hybrid Gametogenesis in Water Frogs (<i>Pelophylax esculentus</i> complex) in the Dniester River Valley (Moldova)

Hybridogenesis is well-known attributes of water frogs of the genus Pelophylax. The P. esculentus complex consists of two parental species, P. lessonae and P. ridibundus, as well as their hemiclonal (sometimes meroclonal) hybrid, P. esculentus. DNA flow cytometry, genetic and morphometric analyses were used to examine water frog compositions in 27 localities (n = 212) throughout the Dniester River valley in Moldova. Two species were revealed in the studied region (42% individuals were P. esculentus and 58% P. ridibundus). All frogs proved to be diploid. We registered the first species in 74% localities and the second in 78%. Populations, where we found only P. ridibundus occurred in 26% localities and only P. esculentus in 22%, while mixed population systems in 52%. Both species were usually observed in open water bodies and rivers that flow down through agricultural land and urban areas. In populations of P. esculentus the number of males strongly exceeded (90%) the number of females. The half of studied hybrid males was sterile, and most of fertile hybrid males produced sperm with the genome of P. lessonae. Several hybrid males gave the mixture of sperm with genomes of P. lessonae and P. ridibundus (amphispermy) and only one male with the P. ridibundus genome. Both species had the mitochondrial DNA of P. ridibundus. Based on nuclear markers, in P. esculentus and P. ridibundus we revealed the presence of alleles of the closely related Anatolian species P. cf. bedriagae. This might affect the successful reproduction of hybridogenetic P. esculentus. However, comparison of percentage of sterile males and alleles of P. cf. bedriagae among hybrids showed no correlation.

Hybrids between Rubus caesius and Rubus sect. Corylifolii (Rosaceae) and their relation to R. cyclomorphus, R. tiliaster, R. glauciformis, R. slesvicensis and R. firmus

Using flow cytometry and microsatellite DNA analysis (MSDA), we studied hybrids between Rubus caesius and various other Rubus species, with an emphasis on Sweden. We show that hybrids between Rubus caesius and Rubus sect. Corylifolii arise easily. They show a large variation in morphology, but can normally be recognised by a number of characters. They are typically hexaploids, but ~13% of the hybrids are pentaploids and ~8% tetraploids. With MSDA, they are harder to identify than hybrids with R. idaeus, partly because all Corylifolii species have themselves arisen from R. caesius hybrids and therefore share many alleles with R. caesius, partly because hybridisation with R. caesius seems to give rise to some variation in microsatellite regions. Hybrids with species of R. sect. Rubus are much rarer in Sweden and we have only identified one such case. We show that R. cyclomorphus and R. tiliaster are not proper apomictic species, at least not in Sweden, but rather a collection of genetically unrelated hybrids between R. caesius and R. raduloides or R. camptostachys. MSDA analysis of R. slesvicensis and R. firmus shows that these species are problematic. We identify two distinct taxa, one from Skåne in Sweden, which we describe as a new species, R. ruboculus and another from the neighbourhood of Viborg and Schleswig, although the latter is probably not identical with R. slesvicensis s.s. All the other samples form a diverse group of putative R. caesius hybrids at three different ploidy levels. Rubus glauciformis is uniform in Småland, Öland and Blekinge, but becomes harder to distinguish from various hybrids in Skåne. We conclude that once R. caesius and R. idaeus hybrids are properly identified, along with a small number of new apomictic species (often with a local distribution), the genus Rubus does not pose any serious taxonomical problems in Sweden.

Exploration and Studies of Wild Cymbopogon martinii (Roxb.) Wats. Growing in Devarayana Durga Hill, Karnataka

Cymbopogon martinii is an aromatic plant commercially grown for its valuable essential oil, which is used in the pharmaceutical and aromatic sectors. Antimicrobial, antifungal, antiviral, anthelmintic, antioxidant, and chemopreventive medicines are all found in the oil. The widespread growth of Cymbopogon martinii was observed in the Devarayana Durga hills of Tumkur, Karnataka, India. The plant was identified using morphological, cytogenetic, and anatomical characteristics (SEM and Microscopic studies). DNA barcoding and flow cytometry analysis was used to confirm ploidy and species specificity. The plant was identified as Cymbopogon martinii (Roxb.) Wats. The seeds and the Herbarium (AC-62/2021) were deposited at the ICAR’s National Bureau of Plant Genetic Resources (NBPGR), Pusa New Delhi. The morphology of the wild plant differed from that of other C. martinii kinds gathered and analyzed from the same wild area, indicating a response to environmental influences. Flow cytometry was used to confirm that the species was tetraploid (C.flexuosus). Using NCBI BLAST, the DNA barcoding sequence of rbcL revealed close similarities to C. martinii, and the sequence was deposited in Gen Bank under the accession number MW538957. As a result, conserving wild C. martinii would aid in the development of new breeding lines with potential bioactive chemicals with an economic worth in the future.

Design, Synthesis and Cytotoxicity Screening of New Thiazole Derivatives as Potential Anticancer Agents through VEGFR-2 Inhibition

Z-configurated isomers are kinetically preferred molecules. Compounds with Z-configuration are contained in many natural products, biologically active compounds and as synthons for organic synthesis. Two series of new thiazole-based analogs were synthesized from appropriate starting materials hydrazinecarbothioamide derivatives (Z)-2a,b to be evaluated for their inhibitory activity towards VEGFR-2. The prepared thiazole compounds 3a-5b were screened for their cytotoxic potency against the MDA-MB-231 breast cancer cell line and their percentage inhibition against VEGFR-2. Compound 4d exhibited good VEGFR-2 inhibitory activity. A DNA flow cytometry analysis was conducted, and compound 4d demonstrated cell cycle arrest at the G1 and G2/M phases of the cell cycle profile and an apoptosis-inducing effect by increasing the percentage of pre-G1 phase. Compound 4d was further evaluated for its apoptosis-inducing effect by studying the effect on mitochondrial membrane potential (MMP) and p53 activation. It was found to boost the level of p53 and reduce the level of MMP compared with the untreated control cells.

Synthesis and Biological Activity Screening of Newly Synthesized Trimethoxyphenyl-Based Analogues as Potential Anticancer Agents.

A group of novel trimethoxyphenyl (TMP)-based analogues were synthesized by varying the azalactone ring of 2-(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxybenzylidene)oxazolone 1 and characterized using NMR spectral data as well as elemental microanalyses. All synthesized compounds were screened for their cytotoxic activity utilizing the hepatocellular carcinoma (HepG2) cell line. Compounds 9, 10 and 11 exhibited good cytotoxic potency with IC50 values ranging from 1.38 to 3.21 μM compared to podophyllotoxin (podo) as a reference compound. In addition, compounds 9, 10 and 11 exhibited potent inhibition of β-tubulin polymerization. DNA flow cytometry analysis of compound 9 shows cell cycle disturbance at the G2/M phase and a significant increase in Annexin-V-positive cells compared with the untreated control. Compound 9 was further studied regarding its apoptotic potential in HepG2 cells; it decreased the level of MMP and Bcl-2 as well as boosted the level of p53 and Bax compared with the control HepG2 cells.

Design, synthesis, and biological evaluation of novel pyrido-dipyrimidines as dual topoisomerase II/FLT3 inhibitors in leukemia cells

Dual inhibition of topoisomerase (topo) II and FLT3 kinase, as in the case of C-1311, was shown to overcome the shortcomings of using topo II inhibitors solely. In the present study, we designed and synthesized two series of pyrido-dipyrimidine- and pseudo-pyrido-acridone-containing compounds. The two series were evaluated against topo II and FLT3 as well as the HL-60 promyelocytic leukemia cell line in vitro. Compounds 6, 7, and 20 showed higher potency against topo II than the standard amsacrine (AMSA), whereas compounds 19 and 20 were stronger FLT3 inhibitors than the standard DACA. Compounds 19 and 20 showed to be dual inhibitors of both enzymes. Compounds 6, 7, 19, and 20 were more potent inhibitors of the HL-60 cell line than the standard AMSA. The results of the in vitro DNA flow cytometry analysis assay and Annexin V-FITC apoptosis analysis showed that 19 and 20 induced cell cycle arrest at the G2/M phase, significantly higher total percentage of apoptosis, and late-stage apoptosis in HL-60 cell lines than AMSA. Furthermore, 19 and 20 upregulated several apoptosis biomarkers such as p53, TNFα, caspase 3/7 and increased the Bax/Bcl-2 ratio. These results showed that 19 and 20 deserve further evaluation of their antiproliferative activities, particularly in leukemia. Molecular docking studies were performed for selected compounds against topo II and FLT3 enzymes to investigate their binding patterns. Compound 19 exerted dual fitting inside the active site of both enzymes.

Apoptosis and Cell Cycle Analysis of the Human Cancer Cell Lines; Breast Michigan Cancer Foundation 7, LS-174T Colon and HePG2 Liver in Response to the Methanolic and Butanolic Extracts ofProsopis juliflora

The phreatophytic tree;Prosopis juliflora(P. juliflora), of the Fabaceae family is a widespread invasive plant. The present study aimed to examine the characteristics of the anti-cancer effects potentially induced byP. julifloraleaves extracts, and to compare the bioactivity of its chemical composition in methanolic and butanolic solvents. MCF-7/breast, LS-174T/colorectal, and HepG2/liver, cancer cell lines were cultivated and incubated with various concentrations of the methanolic and butanolic extracts of the plant leaves, and hence the impact on cell viability, proliferation, and cell cycle stages have been investigated. Both extracts ofP. julifloraleave induced concentration-dependent cytotoxicity against the previously mentioned cancer cell lines. The calculated IC50 averaged 16.7, 18.04 and, 8.10μg/ml for methanolic extract, and 12.5, 32.8 and 5.17μg/ml for butanolic extract ofP. julifloraleaves on MCF-7, LS-174T and, HePG2, respectively. Detailed cellular analyses revealed that the cytotoxic action ofP. julifloraextracts was mainly via apoptosis but not necrosis for MCF7 in both extracts, either. However, apoptosis and necrosis reciprocally detected for LS-174T and HePG2 in response to the methanolic and butanolic extracts, either. The DNA-flow cytometry analysis showed cells specific antiproliferative action and cell cycle stages arrest in the tested cancer cell lines. The antiproliferative action has detected mostly at the G0/G1 rather than the S-phase of the cell cycle in both extracts. For identifying the chemical composition of the plant and evaluating the anticancer potency, both the methanolic and butanolic extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). Betaine and nicotinamide were the major constituents detected for both extracts showing potential anti-cancer effects against breast, hepatic, and colorectal cancerous cells.

Role of DNA Flow Cytometry in the Diagnosis of Malignancy in Bile Duct Biopsies Using Paraffin-Embedded Tissue.

Histopathologic evaluation of bile biopsies for biliary strictures is frequently challenging and is affected by interobserver disagreement. Reliable ancillary tests that can help differentiate benign from malignant are not available. This study aimed to evaluate whether DNA content abnormalities detected by flow cytometry on formalin-fixed, paraffin-embedded (FFPE) tissue can help differentiate benign/reactive, dysplastic from malignant cell populations in bile duct biopsies. We performed DNA flow cytometry on 30 FFPE bile duct biopsies in 5 well-defined diagnostic categories: (1) negative for dysplasia (NED), (2) low-grade dysplasia (LGD), (3) high-grade dysplasia (HGD), (4) carcinoma (CA), and (5) indefinite for dysplasia (IND). Abnormal DNA content was detected in 0 NED, 5 LGD (62.5%), 2 HGD (33.3%), 3 CA (60%), and 4 IND (80%) samples. As a diagnostic marker, the estimated sensitivity, specificity, positive predictive value, and negative predictive value were 63%, 100%, 100%, and 50%, respectively, for diagnosing HGD or CA. DNA flow cytometry analysis is a useful ancillary test for the interpretation of bile duct biopsies. DNA content abnormalities, when correlated with histologic findings, will not only help confirm the morphologic impression but also identify patients who are at a higher risk of developing malignancy.

Hybrids between Rubus idaeus and Rubus sect. Corylifolii and their relation to R. pruinosus and R. rosanthus

We have studied hybrids between Rubus idaeus and various members of R. sect. Corylifolii, primarily in Sweden. With the help of DNA‐ploidy level determinations using flow cytometry and microsatellite DNA analysis of over 500 samples, we show that the material can be divided into four stable apomictic species (belonging to subsect. Subidaei) and a large number of primary hybrids. Stable species can be recognised by a distribution that is distinct from the Corylifolii parent, a uniform morphology and an almost invariant genetic pattern. We confirm that R. cordatiformis has arisen from the hybrid R. eluxatus × idaeus. Moreover, our study shows that R. lagerbergii has arisen from the hybrid R. dissimulans × idaeus. For R. onsalaënsis, we unexpectedly identify the Corylifolii ancestor as a previously unidentified species with a very restricted distribution in the northern part of the province of Halland, which is here described as R. antecedens. Furthermore, we show that R. pruinosus s. str. has arisen from the hybrid R. aureolus × idaeus and that it is known only from a rather restricted area in the provinces of Småland, Östergötland and Södermanland in eastern Sweden. On the other hand, the great majority of what has been called R. pruinosus is a diverse collection of primary hybrids between R. idaeus and various R. sect. Corylifolii species, which show a large variation both genetically and morphologically. The same applies to R. rosanthus, which represents R. norvegicus × idaeus hybrids: The microsatellite analysis shows that they have arisen independently and therefore should be considered as primary hybrids. We also identified primary hybrids between R. idaeus and R. gothicus (by far the most common hybrid in Sweden), R. aureolus, R. camptostachys, R. decurrentispinus, R. eluxatus, R. friesianus, R. hallandicus, R. lidforssii and R. norvegicus.

DNA Flow Cytometry Analysis Relation to Early and Late Clinical Stages in Oral Cancer Patients

DNA Flow Cytometry Analysis Relation to Early and Late Clinical Stages in Oral Cancer Patients

Phytochrome interacting factor proteins regulate cytokinesis in Arabidopsis.

Dicotyledonous plants form an apical hook to protect the fragile apical meristem during upward protrusion from the soil. Etiolated pifq (pif1 pif3 pif4 pif5) seedlings display constitutive apical hook opening. Here, we show that PIF proteins control apical hook opening by regulating the expression of Budding Uninhibited by Benzimidazole 3.1 (BUB3.1) and affecting cytokinesis. Consistent with the major function of BUB3.1 in the organization of phragmoplasts during cytokinesis, the phragmoplasts are well formed in dark-grown pifq but not in wild type. DNA staining and flow cytometry analysis further demonstrate that cellular endoreduplication levels are dramatically reduced in pifq. Chemical treatment with caffeine, an inhibitor of phragmoplast-based cytokinesis, shows that cytokinesis is involved in the apical hook opening. Genetically, BUB3.1 is epistatic to PIFq in the regulation of cytokinesis. Our findings reveal an organ-specific role of PIF proteins in regulating cytokinesis by BUB3.1 during apical hook development.

Design, synthesis and assessment of new series of quinazolinone derivatives as EGFR inhibitors along with their cytotoxic evaluation against MCF7 and A549 cancer cell lines

New acetamide (IV a-e) and 1,3-thiazolidinone derivatives (VII a-e) were designed, synthesized and assessed for their cytotoxic activity against MCF-7 and A549 cell lines along with their lead compounds (erlotinib and gefitinib). The newly designed compounds were prepared according to the adopted procedures in schemes 1 and 2 from their quinazolinone parents. 3D QSAR pharmacophore and docking molecular modeling protocols were conducted using Discovery Studio program, beside a full biological assay for these compounds. The cytotoxicity evaluation demonstrated that compounds IVb, IVc, VIIa, VIIb, VIId exhibited potent cytotoxic activities against both MCF-7 and A549 cell lines. Moreover, the molecular modeling studies corroborated to the affinity of the compounds towards EGFR. Consequently, these five compounds were then screened for their EGFR inhibition and evaluated as well for their toxicity to normal cells, which revealed that the acetamide derivative IVc and the thiazolidinone derivative VIIa were the most potent and least toxic. DNA flow cytometry analysis was conducted for compounds IVc and VIIa, which indicated that they both induced arrest at G2/M phase of the cell cycle.

Abstract 2974: Targeting CD38 inhibits metabolic capacity of acute myeloid leukemia in the tumour microenvironment

Abstract Acute myeloid leukaemia (AML) causes 85,000 global deaths per year, which is estimated to double by 2040. AML has been shown to be highly dependent on the bone marrow (BM) microenvironment. Mesenchymal stromal cells (MSC) specifically, play an important pro-tumoral role. We previously demonstrated AML blasts rely on higher mitochondrial content and are more dependent on oxidative phosphorylation compared to non-malignant unstimulated CD34+ progenitor cells. To achieve this high metabolic demand, functional mitochondria are transferred from the MSC to the AML blasts. Mitochondrial transfer has also been demonstrated in myeloma in a pro-tumoral process regulated by CD38. Taken together, these data indicate that mitochondria are a biologically plausible and attractive drug target in the treatment of AML.We investigated the role mitochondrial transfer and the subsequent metabolic consequences of inhibiting CD38 using daratumumab on the AML BM microenvironment.Primary AML blasts and MSC were isolated from patient's BM. Mitochondrial transfer was assessed by coculture of AML and MSC in vitro using both qPCR DNA analysis and flow cytometry analysis. We used an NSG xenograft mouse model of AML, we transplanted OCI-AML3-luc, and treated the animals with either vehicle control (PBS) or daratumumab (5mg/kg) on day 9 and 16 followed by bioluminescence imaging. Post transplantation, AML mitochondrial transfer was assessed by murine mitochondrial DNA in human AML blasts by species specific PCR analysis. Post transplantation mitochondrial health and function was measured by TMRM and Seahorse analysis.Targeting CD38 using daratumumab inhibits the transfer of mitochondria from MSC to AML in vitro. In vivo, treatment with daratumumab significantly reduced tumor burden and improved survival compared to untreated controls. Additionally, we found two doses of daratumumab resulted in reduced mitochondrial potential, mitochondrial content and oxygen consumption rate in the AML cells sorted from the human xenograft mouse model. Finally, analysis of human AML cells sorted from NSG mouse BM showed reduced levels of mouse mitochondrial DNA in the human AML blasts from daratumumab treated mice compared to mice with AML treated with vehicle control.CD38 inhibition by daratumumab treatment inhibits mitochondrial transfer from MSC to AML blasts in the BM microenvironment. This results in a reduction in oxidative phosphorylation in AML blasts and subsequent reduced leukemia growth, which in turn results in improved NSG/AML animal survival. Whilst it is probable that daratumumab has a number of mechanisms of action, here we demonstrate inhibition of mitochondrial transfer is an addition to the list for this drug in AML. These data support the further clinical investigation of daratumumab based chemotherapeutic strategies as a therapeutic approach for the treatment AML. Citation Format: Jayna J. Mistry, Jamie A. Moore, Christopher R. Marlein, Charlotte Hellmich, Genevra Pillinger, Federica Di Palma, Angela Collins, Kristian M. Bowles, Stuart A. Rushworth. Targeting CD38 inhibits metabolic capacity of acute myeloid leukemia in the tumour microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2974.

Synthesis, Characterization, Anti-proliferative Evaluation, and DNA Flow Cytometry Analysis of Some 2-Thiohydantoin Derivatives.

Due to the well-documented anti-proliferative activity of 2-thiohydantoin incorporated with pyrazole, oxadiazole, quinazoline, urea, β-naphthyl carbamate and Schiff bases, they are noteworthy in pharmaceutical chemistry. An efficient approach for the synthesis of a novel series of 2-thiohydantoin derivatives incorporated with pyrazole and oxadiazole has proceeded via the reaction of the acyl hydrazide with chalcones and/or triethyl orthoformate. Schiff bases were synthesized by the reaction of the acyl hydrazide with different aromatic aldehydes. Moreover, Curtius rearrangement was applied to the acyl azide to obtain the urea derivative, quinazoline derivative, and carbamate derivative. The synthesized compounds structures were discussed and confirmed depending on their spectral data. The anticancer activity of these heterocyclic compounds was evaluated against the breast cancer cell line (MCF-7), where they showed variable activity. Compound 5d found to have a superior anticancer activity, where it has (IC50 = 2.07 ± 0.13 μg/mL) in comparison with the reference drug doxorubicin that has (IC50 = 2.79 ± 0.07 μg / mL). Then compound 5d subjected to further studies such as cell cycle analysis and apoptosis. Apoptosis was confirmed by the upregulation of Bax, downregulation of Bcl-2, and the increase of the caspase 3/7percentage. Insertion of pyrazole, oxadiazole and, quinazoline moieties with 2-thiohydantoin moiety led to the enhancement of its anti-proliferative activity. Hence they can be used as anticancer agents.

Design, synthesis, and cytotoxic screening of novel azole derivatives on hepatocellular carcinoma (HepG2 Cells)

Novel azole derivatives 3–30 were designed, synthesized, and screened for their antitumor activity on HepG2 cell line. The cytotoxicity screening demonstrated that imidazolone 8 and triazoles 25 and 29 exhibited more potent cytotoxic activities by 1.21-, 4.75-, and 1.8-fold compared to Sorafenib (SOR). Furthermore, vascular endothelial growth factor receptor-2 (VEGFR-2) enzyme inhibition assay declared that compounds 25 and 29 had inhibitory activity at the nanomolar concentration. Moreover, the tested compounds exhibited good β-tubulin (TUB) polymerization inhibition percentages. In addition, DNA flow cytometry analysis over HepG2 cells indicated that triazoles 25 and 29 demonstrated arrest at G1 and G2/M phase of the cell cycle and induced apoptotic activity by increasing sub-G1 phase. Finally, mechanistic studies of the proapoptotic activities of compounds 8, 10, 11, 25, and 29 indicated that they induced upregulation of P53, Fas/Fas-ligand, and BAX/BCL-2 ratio expression that resulted in increasing the active caspase 3/7 percentages and trigger apoptosis.