Line HL-60 Research Articles

Assessment of Indicators in a Human Liver Cell Line HL-7702 for Tetracycline Toxicity in Farm Soil

Tetracycline (TC) contamination has become hot research topic, but little attention has been paid to its ecotoxicological monitoring. The primary objective of this study is to investigate the impact of TC on human normal liver cells (HL-7702) and find indicators for monitoring their ecotoxicity. The cytotoxicity of TC, at concentrations ranging from 0 to 1000 μg L−1, was assessed on HL-7702 cells. The results showed that TC significantly inhibited the cell viability at a high concentration (1000 μg L−1). The TC at exposure levels ≥ 50–100 μg L−1 significantly increased the levels of extracellular catalase (CAT), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST), and a significantly positive correlation between the TC concentrations and the values of the above parameters was observed. Swelling of the mitochondrial cristae (MC) and rough endoplasmic reticulum (RER) and the loss of ribosomes in HL-7702 cells, were observed at high TC levels. There was a positive correlation between soil TC concentration and ALT activities. The above results suggest that TC is cytotoxic to HL-7702 cells and that extracellular ALT activities can be used as a sensitive bioindicator for monitoring soil TC contamination. We, therefore, propose that the HL-7702 cell line can be a novel tool for early antibiotics toxicity monitoring.

Toxicological and Nutraceutical Screening Assays of Some Artificial Sweeteners

Artificial sweeteners are food additives worldwide used instead of fructose or glucose in many diet beverages. Furthermore, diet beverages intake has been increasing every year. Thus, some food agencies should regulate it based on toxicological studies. Debates and controversial results are demonstrated, and authority can revise its decision on the basis of new data reporting toxicological effects since cyclamate has been forbidden in some countries. Therefore, the aim of this study was to report new data about the toxicity of acesulfame-k, aspartame, and cyclamate, which are useful for authority agencies, determining the toxic potential and nutraceutical capabilities of these compounds. The toxicity, antitoxicity, genotoxicity, antigenotoxicity, and life expectancy assays were carried out in Drosophila as an in vivo model. In addition, in vitro HL-60 line cell was used to evaluate the chemopreventive activity determining the cytotoxic effect and the capability of producing DNA damage due to internucleosomal fragmentation or DNA strand breaks. Furthermore, the methylated status of these cancer cells treated with the tested compounds was assayed as a cancer therapy. Our results demonstrated that all tested compounds were neither toxic nor genotoxic, whereas these compounds resulted in antigenotoxic and cytotoxic substances, except for cyclamate. Aspartame showed antitoxic effects in Drosophila. All tested compounds decreased the quality of life of this in vivo organism model. Acesulfame-k, aspartame, and cyclamate induced DNA damage in the HL-60 cell line in the comet assay, and acesulfame-k generally increased the methylation status. In conclusion, all tested artificial sweeteners were safe compounds at assayed concentrations since toxicity and genotoxicity were not significantly induced in flies. Moreover, Aspartame and Cyclamate showed protective activity against a genotoxin in Drosophila Regarding nutraceutical potential, acesulfame-k and aspartame could be demonstrated to be chemopreventive due to the cytotoxicity activity shown by these compounds. According to DNA fragmentation and comet assays, a necrotic way could be the main mechanism of death cells induced by acesulfame-k and aspartame. Finally, Acesulfame-K hypermethylated repetitive elements, which are hypomethylated in cancer cells resulting in a benefit to humans.

In Vitro Cytotoxic and Leishmanicidal Activity of Isolated and Semisynthetic ent-Pimaranes from Aldama arenaria.

Two pimaranes ent-pimara-8(14),15-dien-19-oic acid (1: ) and ent-8(14),15-pimaradien-3β-ol (2: ), isolated from Aldama arenaria, and six semi-synthetic derivatives methyl ester of the ent-pimara-8(14),15-dien-19-oic acid (3: ), ent-pimara-8(14),15-dien-19-ol (4: ), acetate of ent-pimara-8(14),15-dien-19-ol (5: ), ent-pimara-8(14),15-dien-19-ol succinic acid (6: ), acetate of ent-8(14),15-pimaradien-3β-ol (7: ), ent-8(14),15-pimaradien-3β-ol succinic acid (8: ) were evaluated in vitro for their cytotoxic activities to childhood leukemia cell lines and leishmanicidal activity against the parasite Leishmania amazonensis. Among these compounds, 1: to 6: presented moderate cytotoxic activity, with compound 4: being the most active (GI50 of 2.6 µM for the HL60 line) and the derivatives 7: and 8: being inactive. Against the parasite Leishmania amazonensis, the most promising derivative was the acetate of ent-pimara-8(14),15-dien-19-ol (5: ), with EC50 of 20.1 µM, selectivity index of 14.5, and significant reduction in the parasite load. Pimarane analogues 1: , ent-pimara-8(14),15-dien-19-oic acid, and 2: , ent-8(14),15-pimaradien-3β-ol, presented different activities, corroborating the application of such molecules as prototypes for the design of other derivatives that have greater cytotoxic or leishmanicidal potential.

METABOLIC PROFILING AND CYTOTOXIC ACTIVITY OF METHANOL EXTRACTS FROM Chamaecrista duckeana (P. BEZERRA & A. FERN.) H. S. IRWIN & BARNEBY (LEGUMINOSAE, CAESALPINIOIDEAE)

The genus Chamaecrista comprises more than 330 species, with only a few studies on their chemical composition and biologic activities. In this study, the phytochemical profile of leaf, stems, and fruits extracts of the C. duckeana were examined by UPLC-ESI-HRMS analysis to determine possibly bioactive constituents. The antioxidant activity was carried out through in vitro assay, by the sequestration of the free radical DPPH. To evaluate the cytotoxic activity of the extracts, an MTT assay was used and the IC50 was determined against HL60 and RAJI cell lines. The metabolic profiles of the botanical parts are dominated by flavonoid class, highlighting isoflavonoids such as daidzin and ononin. All these compounds are reported for the first time in C. duckeana. The extracts presented antioxidant potential, and the activity of the stems extract was higher than the standard butylated hydroxytoluene. In the cytotoxic assay, only HL60 line (leukemia) had growth inhibition over 80%. The stems presented more expressive cytotoxicity with IC50 of 137.3 (104.6-180.1) and 106.8 (96.52-118.3) μmol. L-1 for HL60 and RAJI, respectively. In conclusion, the present work provides an in-depth knowledge about the chemical profile of C. duckeana, a species rich in bioactive secondary metabolites with cytotoxic activity

Abstract P026: NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells

Abstract Background NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine monophosphate (3’-dAMP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. Acute myeloid leukemia (AML) cells exhibit impaired differentiation, uncontrolled proliferation and are highly dependent on mitochondrial metabolic processes for survival and chemoresistance. The aim of this study was to determine the effect of NUC-7738 on oxidative phosphorylation (OXPHOS) proteins and resulting changes in mitochondria morphology and activation of the intrinsic apoptotic pathway. Methods AML cell lines OCI-AML3 and HL-60 were treated with NUC-7738 for 48 hours. Cell differentiation was determined by morphologic modification with Giemsa stain and assessing the expression of surface markers CD11b and CD163 by flow cytometry. Immunoblotting was performed for Mitochondrial Electron Transport Chain Complex I subunit NDUFB8, Complex II subunit SDHB, Complex III subunit UQCRC2, Complex IV subunit COX II and ATP synthase subunit ATP5A. Mitochondria morphology was assessed by confocal microscopy with MitoTracker-Deep Red CMXRos dye. Annexin-V, DAPI, Bcl-2, cytochrome c, and cleaved-caspase 7 were assessed by flow cytometry to determine intrinsic apoptosis. Results NUC-7738 increased the proportion of CD11b+ and CD163+ cells in a dose-dependent manner from 6.1% to 28.7% and 9.6% to 26.9%, respectively. CD11b positivity was also increased in HL-60 cells from 14.5% to 31.1%. NUC-7738 induced morphological differentiation as indicated by decreased nucleo-cytoplasmic ratio, appearance of multi-lobed nuclei and vacuolization of the cytoplasm in OCI-AML3 cells. Decreased nucleo-cytoplasmic ratio was also evident in HL-60 cells. NUC-7738 decreased the protein expression of all 5 complexes of oxidative phosphorylation in OCI-AML3 cells at 48 hours and this metabolic disruption was also evident from distinct changes in mitochondria morphology. NUC-7738 activated the intrinsic apoptotic pathway, causing sequestration of BCL-2 and release of cytochrome c, resulting in a decrease in the percentage of live OCI-AML3 cells from 76% to 37% at 48 hours. Conclusion Through the ability of NUC-7738 to inhibit OXPHOS complexes with consequent terminal differentiation and activation of mitochondrial-mediated apoptosis, it exploits the mitochondrial characteristics of certain cancer cells, including their greater dependence on OXPHOS for survival than normal cells. NUC-7738 offers a potential treatment option for patients with a variety of malignancies. Citation Format: Akbar M. Shahid, In Hwa Um, Oliver J. Read, David J. Harrison. NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P026.

Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer

BackgroundNever in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle. The purpose of this study was to obtain NEK2 protein to prepare an anti-NEK2 monoclonal antibody (mAb) and explore the application of the anti-NEK2 mAb of therapeutic and diagnostic in hepatocellular carcinoma (HCC).ResultsThe NEK2 gene sequence was cloned from the normal liver cell line HL7702, and the full-length NEK2 gene sequence was cloned into the prokaryotic expression vector pET30a and transformed into Escherichia coli BL21 (DE3) cells. The recombinant fusion protein was obtained under optimized conditions and injected in BALB/c mice to prepare an anti-NEK2 mAb. By screening, we obtained a stable hybridoma cell line named 3A3 that could stably secrete anti-NEK2 mAb. Anti-NEK2 3A3 mAb was purified from ascites fluid. The isotype was IgG1, and the affinity constant (Kaff) was 6.0 × 108 L/mol. Western blot, indirect enzyme-linked immunosorbent assay (iELISA), immunofluorescence and immunocytochemical analyses showed that the mAb could specifically recognize the NEK2 protein. MTT assays showed that the mAb 3A3 could inhibit the proliferation of HCC cells. KEGG pathway analysis showed that NEK2 might affected pathways of the cell cycle. Moreover, NEK2-related genes were mainly enriched in the S and G2 phases and might act as tumor-promoting genes by regulating the S/G2 phase transition of HCC cells.ConclusionsAn anti-NEK2 mAb with high potency, high affinity and high specificity was prepared by prokaryotic expression system in this study and may be used in the establishment of ELISA detection kits and targeted treatment of liver cancer.

Design, Synthesis and Anticancer Activity of a New Series of N-aryl-N'-[4-(pyridin-2-ylmethoxy)benzyl]urea Derivatives.

The development of cancer treatments requires continuous exploration and improvement, in which the discovery of new drugs for the treatment of cancer is still an important pathway. In this study, based on the molecular hybridization strategy, a new structural framework with an N-aryl-N’-arylmethylurea scaffold was designed, and 16 new target compounds were synthesized and evaluated for their antiproliferative activities against four different cancer cell lines A549, MCF7, HCT116, PC3, and human liver normal cell line HL7702. The results have shown seven compounds with 1-methylpiperidin-4-yl groups having excellent activities against all four cancer cell lines, and they exhibited scarcely any activities against HL7702. Among them, compound 9b and 9d showed greatly excellent activity against the four kinds of cells, and the IC50 for MCF7 and PC3 cell lines were even less than 3 μM.

Effect of the reactive oxygen species-induced by bisphenol A on liver lipid metabolism disorder

To explore the toxic effect of bisphenol A on the liver, as well as the influence effect on lipid metabolism. The toxic effects of bisphenols on human health were studied by using in vivo experiments of bisphenol A exposure in rats and in vitro experiments of human liver cell line HL-7702. Male SD rats were divided into control group(Ctrl), 1 mg/(kg·d) group(low), 5 mg/(kg·d) group(medium) and 25 mg/(kg·d) group(high) for 14 days subacute exposure of bisphenol A, to evaluate the toxic effect of bisphenol A on the liver in terms of body weight, liver organ index, liver pathological tissue sections, serum biochemical indicators. Then HL-7702 was divided into four groups: control group(Ctrl), low concentration treatment group(0. 16 μmol/L), medium concentration treatment group(4 μmol/L) and high concentration treatment group(100 μmol/L). After 24 hours of exposure to bisphenol A, the contents of triglyceride(TG) and total cholesterol(TC) in cells, reactive oxygen species(ROS) levels were detected, and the transcription levels of genes related to lipid metabolism and oxidative stress were detected by fluorescent quantitative PCR. The 14-day subacute exposure had no significant effect on rat body weight and liver body weight ratio, but liver pathological sections clearly showed that bisphenol A exposure can damage liver tissue structure. Serum biochemical indicator of total bile acid(TBA) was significantly reduced in the high-dose group, which was(4. 75±0. 33)μmol/L, creatinine(Cr) was significantly increased in the medium and high-dose group, which were(18. 00±0. 76)μmol/L and(18. 83±0. 75)μmol/L, respectively. TC, high-density lipoprotein(HDL-C) and low-density lipoprotein(LDL-C) were significantly reduced in the middle-and high-dose groups(P<0. 05), which were(1. 44±0. 10), (1. 14±0. 10)mmol/L;(0. 84±0. 04), (0. 63±0. 07)mmol/L and(0. 21±0. 04), (0. 16±0. 05)mmol/L, respectively. Bisphenol A exposure could significantly reduce the content of TC in hepatocytes(P<0. 05). BPA treatment could significantly increase ROS levels in HL-7702 cells. The transcription level of PPARα was significantly increased in the high concentration group, FABP1 was significantly increased in the high concentration group, SOD1 was significantly decreased in the medium and high concentration group(P<0. 05). Bisphenol A may cause oxidative stress by inducing excessive ROS production in liver cells, leading to liver damage and disorder of lipid metabolism in the body, thereby showing liver toxicity.

QSAR Classification of Anticancer Heterocyclichydrazones With Reactivity Descriptors

In this study, validated PLS-DA models that discriminate heterocyclichydrazones with potent anticancer activity (GI50/IC50<50x10-6M) from those with low activity (GI50/IC50>=50x10-6M) against the cancer cell lines HCT-116 (colon), OVCAR-8 (human ovary), HL-60 (leukemia), and SF-295 (glioblastoma) were developed. A dataset of 24-a-(N)-heterocyclichydrazones and 14 N-acylhydrazonyl-thienyl and various global and local reactivity descriptors were used for modeling. The best models classified for training and test sets with an accuracy range between 67-100%, a class specificity and sensitivity range between 71-100%, error rate range between 0-0.27, and non-error rate range between 0.73-1.0. An external set of 20 compounds was predicted and the models showed which new compounds are not suggested for further biological investigation. The molecular properties with impact on the modeled endpoints show that the antitumor activity can be improved with electron-acceptor N-acylhydrazonyl-thienyl derivatives and a-(N)-heterocyclichydrazones with moderate electron-donating character.

Evaluation of the cytotoxicity of products obtained from Calotropis procera (Apocynaceae)

Calotropis procera belongs to the Apocynaceae family and is found in the Northeast of Brazil. Several activities are attributed to this species, such as anti-inflammatory, antibacterial and anti-cancer activities. This research aimed to conduct a bioguided study of C. procera in order to isolate and identify triterpenes of the species and evaluate the cytotoxic activity of products of the plant species. The crude ethanolic extract (CEE) was obtained and partitioned. The hexanic phase was chromatographed giving the compound Cp-1, which was identified using spectroscopic techniques and thermal analysis. It was possible to identify Cp-1 as a triterpene called calotropenyl acetate, already reported in the literature. The cytotoxicity test was performed using the MTT technique and it was observed a good activity of CEE and hexanic phase against HL-60 and KS62 lines with an IC50 ranging between 26.8 ± 0.4 µg mL-1 and 47.0 ± 0.5 µg mL-1, besides no toxicity in normal lines, while Cp-1 showed no relevant activity against the tumor cells tested. The results indicated that the species contains in its chemical composition products with pharmacological interest, in addition to cytotoxic for leukemic cells, which can be further explored in complementary studies.

New cytotoxic zinc(II) and copper(II) complexes of Schiff base ligands derived from homopiperonylamine and halogenated salicylaldehyde

The zinc(II) and copper(II) complexes of three new Schiff base ligands (H-La, H-Lb, H-Lc) derived from homopiperonylamine and the respective halogenated salicylaldehyde, [Zn(La)2] (1), [Zn(Lb)2] (2), [Cu(Lc)2] (3), were synthesized and fully characterized by means of IR, ESI-MS, elemental analysis and X-ray single crystal diffraction analysis. The complexes 1–3 exhibited significantly higher anti-proliferation activities than the corresponding ligands against the tumor cell lines T-24, HepG2 & SK-OV-3, but without obvious cytotoxicity on normal liver cell line HL-7702. Especially, complex 2 could induce cell cycle arrest of HepG2 cells in S phase and also cell apoptosis through mitochondria-related apoptotic pathway. DNA binding studies supported by spectral analysis, DNA viscosity measurement and gel electrophoresis suggested that complexes 1–3 most likely bound with DNA in an intercalative mode, while the electrostatic interactions existed between complex 1 or 2 and DNA might be also considered.

DNA damage of human derived liver cell line HL-7702 induced by organic extracts from surface water in Pearl River Delta, China.

Many organic pollutants attract public health concern due to their genotoxicity. To investigate the genotoxicity of organic matter in surface water of the Pearl River Delta (PRD). Organic substances of 24 samples (dry and wet season) from North River, West River and East River were extracted from 60 L source water by XAD-2 macroporous resin. DNA damage effect of organic extracts was tested in human derived liver cells (HL-7702), using single cell gel electrophoresis (SCGE) assay. The results showed that 100% organic extracts (24/24) could induce DNA damage in HL-7702 cells when the concentration was above 1.0 L surface water/ml culture, no significant difference of DNA damage between dry and wet seasons was observed. The organic substance-induced DNA damage in HL-7702 cells was significantly (P < 0.05) correlated with the contents of Dissolved Organic Carbon in both seasons and Total Suspended Solids in dry season. In conclusion, organic extracts induced genetic damage in HL-7702 cells, indicating potential genotoxicity of organic pollutants of surface water from PRD, South China.

MiR-221-3p promotes hepatocellular carcinogenesis by downregulating O6-methylguanine-DNA methyltransferase

ABSTRACT This study aimed to investigate the influence of miR-221-3p and O6-methylguanine-DNA methyltransferase (MGMT) interaction in human hepatocellular carcinoma (HCC), thereby revealing a novel molecular mechanism of hepatic carcinogenesis involving miR-221-3p and MGMT. Fluorescence qPCR and immunoblot assays were performed to determine the expression of RNA and protein in HCC tissues and cell lines. We also employed the firefly and Renilla luciferase assay to verify the target relationship between miR-221-3p and MGMT mRNA. Assessments including the MTT assay, wound-healing assay, transwell assay, colony foci formation experiment, and flow cytometric experiment were carried out to determine the viability, migration, invasion, proliferation, cell cycle progression, and apoptosis of SMMC-7721 and BEL-7404 cell lines with the modulated expression of miR-221-3p and MGMT. Compared to healthy tissues and cell line HL7702, miR-221-3p was significantly upregulated but MGMT was significantly downregulated in carcinomas and cancerous cell lines. Forced miR-221-3p overexpression was found to enhance the proliferation, migration, invasion, and clonogenicity of cell lines, but it suppressed cell apoptosis. Findings also revealed that forced miR-221-3p overexpression had little effect on cell cycle progression. After MGMT was confirmed to be atarget gene of miR-221-3p, it was found that the forced upregulation of miR-221-3p downregulated MGMT mRNA and protein levels significantly. MiR-221-3p was identified as an HCC promoting factor, and it specifically inhibited the expression of the MGMT. Besides, the upregulation of miR-221-3p had apositive influence on HCC pathogenesis by inhibiting MGMT expression.

Long Non-Coding RNA NRAD1 and LINC00152 are Highly Expressed and Associated with Prognosis in Patients with Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma (HCC) is prevalent throughout the world. The aim of this study is to explore new long non-coding RNAs (lncRNAs) associated with hepatocellular carcinoma and detect their expression levels in hepatocellular carcinoma cell lines and tissues. These results will provide new clues on further function and biomarker studies of HCC-related lncRNAs.Patients and MethodsAll patients were diagnosed as HCC between 30th, March, 2015 and 30th, July, 2018. LncRNA human gene expression microarray was applied to the profiling of lncRNAs in four cancerous tissues and the paired paracancerous tissues.ResultsWe retrospectively reviewed 63 patients with primary HCC who underwent a curative liver resection at the Department of Hepatology, Qingdao Sixth People’s Hospital. The expression level of lncRNA NRAD1 and LINC00152 was detected by real-time PCR. Prognostic factors were evaluated using Kaplan–Meier curves and Cox proportional hazards models. By microarray profiling of lncRNAs, 256 lncRNAs were found to be differentially expressed, including 162 upregulated and 94 downregulated (P<0.05, fold change>2). Two candidate lncRNAs were determined as the targets in this study, which were NRAD1 (upregulated by 6.35 fold), LINC00152 (upregulated by 4.53 fold). NRAD1 and LINC00152 were downregulated in the normal liver cell lines Chang liver, HL7702, THLE-2, THLE-3, FL62891 and AML12, which were significantly lower than HCC cell lines SMMC-7721, Hep3B, HuH7, MHCC-97H, HCC-LM and SK-Hep-1 (P<0.05). Overexpression of lncRNA NRAD1 and LINC00152 was associated with decreasing OS rates, respectively (P=0.0263 and P=0.0285). Meanwhile, overexpression of NRAD1 and LINC00152 was associated with decreasing PFS rates, respectively (P=0.0174 and P=0.0041). After adjusting for competing risk factors, we identified that microvascular invasion (P=0.014), tumor size (P=0.026), lncRNA NRAD1 (P=0.001) and LINC00152P9 (P=0.036) expression levels were independent prognostic factors associated with prognosis of patients with HCC.ConclusionWe found lncRNA NRAD1 and LINC00152 expressed significantly higher in HCC tissues compared with non-tumorous tissues. Overexpression of lncRNA NRAD1 and LINC00152 were independent risk factors associated with the prognosis of patients with HCC.

The copper(II) complexes of new anthrahydrazone ligands: In vitro and in vivo antitumor activity and structure-activity relationship

Two new copper(II) complexes, 9-PMAH-Cu (1) and 9-FPMAH-Cu (2), of anthrahydrazone were synthesized and structurally characterized, in which 9-FPMAH (9-(4′-trifluoromethyl)-pyrimidine anthrahydrazone) is the 4′-CF3 derivative of 9-PMAH (9-pyrimidine anthrahydrazone). Both complexes 1 and 2 showed similar intercalative binding modes towards DNA and might compete with the typical DNA intercalator, GelRed, in the same binding site. They could also act as topoisomerase (type I) suppressor to effectively inhibit its activity, in which complex 1 was more effective than 2. The in vitro antitumor screening indicated that complex 1 displayed much higher antiproliferative ability than 2 and cisplatin towards all the tested tumor cell lines. On the other hand, complex 1 also showed high cytotoxicity against human normal liver cell line HL-7702, suggesting it is a potential high cytotoxic antitumor candidate. While it was also suggested that the loss of activity of complex 2 might be due to the presence of 4’-CF3 on the pyrimidine ring. Studies on the cellular level showed that complex 1 could arrest the cell cycle of the most sensitive T-24 cells at G2/M phase and induced cell apoptosis. Complex 1 further showed a significant suppression on the tumor growth on the T-24 tumor xenograft mouse model, but not reduced the body weight. Especially, complex 1 could retain its coordination state in H2O even in the presence of HSA. The results suggests that complex 1 is of enough safety to be considered as a promising anticancer candidate by combining the bioactive Cu(II) and the anthrahydrazone pharmacophore.

Synthesis and anti-tumor properties of derivatives [4- (41-chlorophenyl)-5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[c,d]azulen-1-yl-metil]-para-tolylamine

Leukemia, as a part of hemoblastosises, is a malignant blood system disease, which is characterized by bone marrow damage, caused by leukemic stem cells, which appear due to disruption of self-renewal and differentiation of hempoetic stem cells and predecessor cells. In their turn, hemoblastoses are divided into two groups: bone marrow (acute leukemia, chronical leukemia, paraproteinemic hemoblastoses) and outside bone marrow (lymphogranulomatosis, or Hodgkin lymphoma, and non-Hodgkin malignant mymphomas).&#x0D; Nowadays in Ukraine, different kinds of leukemia are cured by various drugs, which have many side effects. Increase in effectivity of chemotherapy of tumor disease is primarily related to creation of new antitumor drugs of selective action. Which is why search for biologically active compounds with antitumor activity is a perspective direction in creation of new drugs.&#x0D; Aim of this work was synthesis of compounds with potential antitumor properties in a variety of [4-(41-chlorophenyl)-5,6,7,8-tetrahydro-2,2a,8a-triazacyclopenta[c,d]-azulen-1-yl-methyl]-para-tolylamin derivatives.&#x0D; As the objects of our studies, we have picked the derivatives of [4-(41-chlorophenyl)-5,6,7,8-tetrahydro-2,2а,8а-triazacyclopenta[c,d]-azulen-1-yl-methyl]-para-tolilamin (8 and 10 a, b). [4-(41-Chlorphenyl)-5,6,7,8-tetrahydro-2,2а,8а-triazacyclopenta[c,d]azulen-1-yl-methyl]-para-tolylamin (8) was obtained by boiling of equimolar quantities of 3-(41-methylphenyl)aminomethyl-6,7,8,9-tetrahydro-5Н-[1,2,4]triazolo[4,3-a]azepin (5) and α-brom-4-chloracetophenon in ethylacetate. Thioamides (10 a, b) were obtained by interaction of amin (8) with corresponding arylisothiocyanates (9 а, b) in dry benzene.&#x0D; Antitumor activity of [4-(41-chlorphenyl)-5,6,7,8-tetrahydro-2,2а,8а-triazacyclopenta[c,d]azulen-1-yl-methyl]-para-tolylamin (8) was studied in National Cancer Institute of Health, USA within Development Therapeutic Program.&#x0D; In experimental conditions [4-(41-chlorphenyl)-5,6,7,8-tetrahydro-2,2а,8а-triazacyclopenta[c,d]azulen-1-yl-methyl]-para-tolylamin (8) showed ability to inhibit growth of cancerous leukemia cells of CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226 and SR lines, higher than standard – 5-fluorouracil. Towards HL-60(TB) cells [4-(41-chlorphenyl)-5,6,7,8-tetrahydro-2,2а,8а- triazacyclopenta[c,d]azulen-1-yl-methyl]-para-tolylamin exceeds standard in effectivity by 64.68%. For K-562, MOLT-4, RPMI-8226 and SR cells, those numbers are equal to: 85.88%, 84.95%, 42.10% and 36.82% correspondingly. Towards CCRF-CEM cells, this compound not only inhibits cell growth and division, but also destroys them by 20.34%.&#x0D; Thus conducted studies confirm perceptivity of search for compounds with antitumor action on the basis of [4-(41-chlorophenyl)-5,6,7,8-tetrahydro-2,2а,8а-triazacyclopenta[c,d]azulen-1-yl­methyl]-para-tolylamin.

Exosomes function as nanoparticles to transfer miR-199a-3p to reverse chemoresistance to cisplatin in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a frequently seen malignant tumor globally. The occurrence of cisplatin (DDP) resistance is one of the main reasons for the high mortality of HCC patients. Therefore, it is of great theoretical significance and application value to explore the mechanism of chemotherapy resistance. Drug resistance can be modulated by exosomes containing mRNAs, micro RNAs (miRNAs) and other non-coding RNA (ncRNAs). Exosomal miR-199a-3p (Exo-miR-199a-3p) was subjected to extraction and verification. Whether exo-miR-199a-3p could make HCC cells sensitive to DDP in vitro was verified via flow cytometry, Cell Counting Kit-8 (CCK-8) assay, immunofluorescence assay and Transwell assay. Intravenous injection of exo-miR-199a-3p and intraperitoneal injection of DDP were carried out in vivo. Moreover, the possible targets of miR-199a-3p were screened through bioinformatics analysis, which were ascertained by Western blotting (WB). Then, miR-199a-3p levels in human normal liver epithelial cell line HL-7702 and HCC cell lines HuH7 and HuH7/DDP were elevated in a concentration-dependent manner. Exo-miR-199a-3p has abilities to adjust underlying targets and conjugate cells, to repress cells to invade, stimulate their apoptosis and abate their ability. Additionally, the caudal injection of exo-miR-199a-3p reversed the chemoresistance of tumors and slowed down their growth in the body owing to the up-regulation of miR-199a-3p and down-regulation of underlying target proteins in tumors. Finally, exo-miR-199a-3p was found to overturn the HCC’s resistance to DDP, and it may function in DDP-refractory HCC therapy as an underlying option in the future.

Over-expression of miR-144 inhibits invasion of liver cancer SMMC-7721 cells in vitro by suppressing TLR/MyD88 pathway

To investigate the effects of over-expression of miR-144 on invasion of SMMC-7721 cells and Toll-like receptor (TLR)/myeloid differentiation factor 88 (MyD88) pathway in hepatocellular carcinoma cells. The expressions of miR-144 was examined in normal human hepatocyte line HL-7702 and hepatocarcinoma cell line SMMC-7721 using realtime quantitative PCR (qRT-PCR). SMMC-7721 cells were divided into blank group, miR-144 NC group and miR-144 mimics group, and the expressions of miR-144 in each group were detected with qRT-PCR. Cell count kit-8 (CCK8) was used to assess the survival of SMMC-7721 cells, and the cell invasion was evaluated using Transwell assay. The expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and TLR/MyD88 pathway-related proteins in the cells were detected with Western blotting; the effect of 40 μ mol/L MyD88 inhibitor on TLR/MyD88 pathway-related proteins was examined in SMMC-7721 cells. Compared with normal human hepatocytes, SMMC-7721 cells expressed a significantly lower level of miR-144 (P &lt; 0.05). CCK-8 assay showed that test showed that miR-144 over-expression significantly decreased the cell survival rate (P &lt; 0.05), lowered the number of invasive cells, and decreased the expression of MMP-2 and MMP-9 in SMMC-7721 cells (P &lt; 0.05). The expressions of Toll-like receptor 4 (TLR4), MyD88, phosphorylated nuclear factor-kappa B (pNF-κB) and NF-κB protein decreased significantly in miR-144 mimics group and TJ-M2010-2 group (P &lt; 0.05) and were comparable between the two groups (P &gt; 0.05). Overexpression of miR-144 decreases SMMC-7721 cell survival and invasion by inhibiting TLR/MyD88 pathway.

Cytotoxicity of the Sesquiterpene Lactone Spiciformin and Its Acetyl Derivative against the Human Leukemia Cell Lines U-937 and HL-60.

Spiciformin (1) is a sesquiterpene lactone with a germacrane skeleton that is found in two Tanacetum species endemic to the Canary Islands. In this study, the cytotoxicities of 1 and its acetyl derivative (2) were evaluated against human tumor cells. These sesquiterpene lactones were cytotoxic against human acute myeloid leukemia (U-937 and HL-60) cells, even in cells over-expressing the pro-survival protein Bcl-2, but melanoma (SK-MEL-1) and human mononuclear cells isolated from blood of healthy donors were more resistant. Both compounds are apoptotic inducers in human leukemia U-937 cells. Cell death was mediated by the processing and activation of initiator and effector caspases and the cleavage of poly(ADP-ribose) polymerase, and it was blocked by a broad-spectrum caspase inhibitor and (in the case of sesquiterpene lactone 2) by the selective caspase-3/7, -8, and -9 inhibitors. In addition, certainly in the case of compound 2, this was found to be associated with a decrease in mitochondrial membrane potential, downregulation of the anti-apoptotic protein Bcl-2, activation of the mitogen-activated protein kinases signaling pathway, and generation of reactive oxygen species. It will, therefore, be relevant to continue characterization of this class of compounds.

JNK pathway promotes hepatocyte apoptosis by inhibiting Bcl-2 and upregulating expressions of Bim, caspase-3 and caspase-9 after cardiopulmonary bypass

Purpose: To study the effect of Jun N-terminal kinase (JNK) signaling pathway on hepatocyte apoptosis in vivo and in vitro, and to elucidate the mechanism of action.&#x0D; Methods: TdT-mediated dUTP Nick-End Labeling (TUNEL) method was used to determine apoptosis in control and cardiopulmonary bypass (CPB) groups at 0, 3 and 6 hours after rat surgery. The expressions of JNK and p-c-Jun in liver tissues at 0, 3 and 6 h after surgery, and the levels of p-c-Jun, Bcl-2 and Bim following overexpression of JNK, were determined using Western blot assay. Human liver cell line HL-7702 was cultured and transfected with over-expressed JNK plasmid and empty plasmid. Proliferation of HL-7702 cells after JNK over-expression was assessed by Cell Counting Kit-8 (CCK-8), while quantitative real-time polymerase chain reaction (RT-qPCR) was employed to evaluate mRNA expression levels of caspase-3 and caspase-9 mRNA after JNK over-expression. Apoptosis of the cells was determined by flow cytometry (FC) after JNK over-expression.&#x0D; Results: FC results showed that the number of apoptotic hepatocytes increased after JNK overexpression in hepatocytes while TUNEL assay results demonstrated that hepatocyte apoptosis increased in CPB group, when compared to control group; furthermore, the number of apoptotic cells gradually increased within 6 h after surgery. The expressions of JNK and p-c-Jun were higher in CPB group than in control group, and increased gradually in both groups within 6 h after surgery. Overexpression of JNK decreased the proliferation of hepatocytes, and also lowered protein expression levels of p-c-Jun and Bim; on the other hand, the protein expression levels of Bcl-2 fell, while mRNA expression levels of caspase-3 and caspase-9 mRNA increased.&#x0D; Conclusion: JNK pathway promotes hepatocyte apoptosis after cardiopulmonary bypass by inhibiting Bcl-2 pathway and promoting the expressions of Bim caspase-3 and caspase-9.&#x0D; Keywords: Cardiopulmonary bypass, Apoptosis, JNK pathway, Bim, caspase-3 and caspase-9