Acetylshikonin (AcSh), as a red colored pigment found in roots of the plants from family Boraginaceae, showed excellent cytotoxic activity. Due to its hydrophobic nature, and thus poor ...bioavailability, the aim of this study was to prepare acetylshikonin/β-cyclodextrin (AcSh/β-CD) inclusion complex by using coprecipitation method, characterize obtained system by using UV/VIS, IR and 1H NMR spectroscopy, and determine cytotoxic activity. Phase solubility test indicated formation of AL-type binary system (substrate/ligand ratio was 1:1 M/M), with stability constant Ks of 306.01 M−1. Formation of noncovalent bonds between inner layer of the hole of β-CD and AcSh was observed using spectroscopic methods. Notable changes in chemical shifts of two protons (−0.020 ppm) from naphthoquinone moiety (C6-H and C7-H), as well as protons from hydroxyl groups (−0.013 and −0.009, respectively) attached to C5 and C8 carbons from naphthoquinone part indicate that the molecule of AcSh enters the β-CD cavity from the aromatic side. Cytotoxic activity against HCT-116 and MDA-MB-231 cell lines was measured by MTT test and clonogenic assay. Mechanisms of action of free AcSh and inclusion complex were assessed by flow cytometry. In comparison to free AcSh, AcSh/β-CD showed stronger short-term effect on HCT-116 cells and superior long-term effect on both cell lines. Inclusion complex induced more pronounced cell cycle arrest and autophagy inhibition, and induced increase in accumulation of intracellular ROS more effectively than free AcSh. In conclusion, AcSh/β-CD binary system showed better performances regarding cytotoxic activity against tested tumor cell lines.
Chronic lymphocytic leukemia (CLL) develops due to an imbalance between apoptosis and proliferation of B lymphocytes. Chrysin induced apoptosis in leukemia cell lines such as U937, MO7e, THP-1 and ...HL-60, but there has not yet been data demonstrating the apoptotic effect of chrysin on CLL cells. Therefore, in our investigation we examined the cytotoxicity of chrysin against two leukemia cell lines, MOLT-4 and JVM-13, peripheral blood lymphocytes isolated from B-CLL patients and peripheral blood mononuclear cells (PBMC) from healthy individuals in vitro. The effect of chrysin on viability of MOLT-4 and JVM-13 cell lines, B-CLL cells derived from 28 patients and PBMC from 16 healthy subjects was determined by MTT assay. The type of cell death induced by chrysin was verified by Annexin V/7AAD assay and acridine orange and ethidium bromide (AO/EB) staining assay. Intracellular localisation and endogenic expression of apoptotic proteins including Bax, Bcl-2, cytochrome c and caspase-3 were determined by flow cytometry and fluorescent microscopy. Our results demonstrated that exposure of MOLT-4, JVM-13 cell lines and B-CLL cells to the concentration of chrysin of 10μM and higher selectively decreased viability of cells in this cell population, but not in the PBMC derived from healthy subjects; LC50 values of chrysin for B-CLL cells were 51μM for 24 hours and 32μM for 48 hours of incubation, respectively. Our findings demonstrated that chrysin induces the activation of proapoptotic Bax and decreases the expression of antiapoptotic Bcl-2 protein, releases cytochrome c from mitochondria into cytosol and cleavages/activates caspase-3, subsequently leading to the activation of apoptosis of B-CLL cells. Together, these findings suggest that chrysin selectively induces apoptosis of peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia patients via mitochondrial pathway in vitro and that it might have a promising role as a potential future antileukemic remedy.
Background/Aim. The main cause for development of endometrial hyperplasia is unopposed effect of estrogen on endometrial cells. The aim of our study was to investigate and compare cytotoxic and ...apoptotic effects of mifepristone on human endometrial stromal cell line for the first time. Both percentage of cytotoxic and apoptotic cells were determined after 24 h treatment with different doses of mifepristone. Methods. The percentage of cytotoxic cell was evaluated by viability assay while the percentage of apoptotic cells was determined using flow cytometry. Determination of apoptotic effects was confirmed using immunofluorescence method determining expression and localization of active Bax and Bcl-2 proteins. Results. Our results indicated that mifepristone induced cytotoxic and apoptotic effect on human endometrial stromal cell line (ThESC) through changes in expression level of Bcl-2 and active Bax proteins. Conclusion. Cytotoxic and pro-apoptotic effects of mifepristone on human endometrial stromal cell line in vitro was investigated in this study for the first time. It is crucial to point out that mifepristone expressed both cytotoxic and pro-apoptotic effect on ThESC cell line. Our results may contribute to determination of localization and expression level of the crucial proteins involved in apoptosis in ThESC cell line after the treatment with the lowest cytotoxic doses of mifepristone.
Endometrial hyperplasia is a condition that occurs as a result of hormonal imbalance between estrogen and progesterone. Morphological disturbance of endometrial cells occurs consequently leading ...towards endometrial cancer. In therapy of endometrial hyperplasia SERMs are used to supress effects of locally high estrogen level in uterus. There is strong evidence suggesting that estrogen could be involved in cell death - apoptosis. There are no experimental data demstrating the direct apoptotic effect of both raloxifene and estrogen on the ThESC cell line. The aim of our study wa sto investigate both cytotoxic and apototic mechanism of raloxifene and estrogen - induced death in the ThESC cell line.
In order to determine their cytotoxic and apoptotic effects, various doses of raloxifene and estrogen were applied to the ThESC cell line for 24 h. After the treatment MTT assay, FACS analysis and immunofluoroscence method were conducted.
The results of this study for the first time demonstrated the cytotoxic and apoptotic effects of raloxifene and estrogen on human endometrial stromal cell line suggesting the involvement of the inner, mitochondrial apoptotic pathway.
Our results demonstrated apoptotic effects of investigated drugs in the ThESC cell line through increasing the Bax/Bcl-2 ratio and activation of caspase 3.
Background/Aim. Chronic lymhocytic leukemia (CLL) is considered more as a disease of cells accumulation due to the defect in apoptosis rather than deregulated cell?s proliferation. The activation of ...apoptosis is one of the main molecular mechanisms responsible for anti-cancer activities of most of the currently studied potential anti-cancer agents, including natural compounds. Teucrium polium (TP) extracts exhibited strong cytotoxic effects in murine leukemia cell line, RAW 264.7 and human melanoma cell line, C32, but their cytotoxic effects against human leukemia cells were unknown. Methods. The viability of human leukemia cell lines (MOLT 4 and JVM 13), lymphocytes isolated from 28 patients with CLL (CLL cells), and peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy subjects treated with TP leaves methanolic extract, was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis of TP treated CLL cells was measured by flow cytometry applying Annexin V/7AAD staining. The expressions of active proapoptotic protein Bax, antiapoptotic protein Bcl-2, cytochrome c and the percentage of cells containing cleaved caspase-3 in treated CLL cells was determined by flow cytometry and immunocytochemistry. Results. The TP meth-anolic extract decreased the viability of all tested human leukemia cells but it had no effect on the viability of PBMCs isolated from healthy subjects. The cytotoxic effect of TP was caused by its induction of CLL cells? apoptosis. TP disarranged the ratio of the expressions of proapoptotic Bax and antiapoptotic Bcl-2 protein in favor of Bax, consequently inducing apoptosis by cytochrome c mitochondrial release and activation of caspase-3 in treated CLL cells. Conclusion. The TP leaves methanolic induced selective apoptosis in CLL cells and it affected the expressions of key proteins involved in the regulation of programmed cell death.
New twelve in silico designed coumarin-based ERα antagonists, namely 3DQ-1a to 3DQ-1е, were synthesized and confirmed as selective ERα antagonists, showing potencies ranging from single-digit ...nanomolar to picomolar. The hits were confirmed as selective estrogen receptor modulators and validated as antiproliferative agents using MCF-7 breast cancer cell lines exerting from picomolar to low nanomolar potency, at the same time showing no agonistic activity within endometrial cell lines. Their mechanism of action was inspected and revealed to be through the inhibition of the Raf-1/MAPK/ERK signal transduction pathway, preventing hormone-mediated gene expression on either genomic direct or genomic indirect level, and stopping the MCF-7 cells proliferation at G0/G1 phase. In vivo experiments, by means of the per os administration to female Wistar rats with pre-induced breast cancer, distinguished six derivatives, 3DQ-4a, 3DQ-2a, 3DQ-1a, 3DQ-1b, 3DQ-2b, and 3DQ-3b, showing remarkable potency as tumor suppressors endowed with optimal pharmacokinetic profiles and no significant histopathological profiles. The presented data indicate the new compounds as potential candidates to be submitted in clinical trials for breast cancer therapy.
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•Twelve novel coumarin-based ERα antagonists, 3DQ-1a to 3DQ-1е, were synthesized•Compounds exerted picomolar to nanomolar potency against ERα and MCF-7 cell lines•Compounds acted at Raf-1/MAPK/ERK, hERE and AP-1, and MCF-7 G0/G1 phase levels•Compounds suppressed breast cancer in vivo showing optimal pharmacokinetic profiles•3DQ-4a, 3DQ-2a, 3DQ-1a, 3DQ-1b, 3DQ-2b, and 3DQ-3b are clinical trials candidates
Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. Although the local application of low doses of methotrexate (MTX) is used as an effective treatment of ...the myomas, myotrexate could be a promising new drug. This study investigated the cytotoxic and apoptotic effects of both MTX and myotrexate in human fibroblasts derived from the uterine fibroids (T hES cell line). The myotrexate adduct is an aqueous solution of MTX and L-arginine. Cells were treated with a graded concentrations of both MTX and myothrexate (0.1-16 µM) for 24 h. The cytotoxicity was assayed by MTT test, apoptosis was evaluated by Annexin V-FITC assay and their possible role in apoptosis was determined by immnu- flourescence. Both MTX and myotrexate induced apoptosis in T hES cells in a dose dependent manner (p < 0.001). Myotrexate significantly increased the percentage of AnnexinV positive cells, BAX/Bcl-2 ratio and subsequent caspase-3 activation compared to the MTX treated cells (p < 0.05). Both MTX or myotrexate treatment showed a diffuse staining of cytochrome c indicating its release from mitochondria to the cytosol, suggesting that their mechanisms of action most likely involves the mitochondrial apoptotic pathway.
Background & Aims We used Concanavalin A-induced liver injury to study the role of Interleukin 33 and its receptor ST2 in the induction of inflammatory pathology and hepatocellular damage. Methods We ...tested susceptibility to Concanavalin A induced hepatitis in ST2 deficient and wild type BALB/c mice and analyzed the effects of single injection of Interleukin 33 as evaluated by liver enzyme test, quantitative histology, mononuclear cell infiltration, cytokine production, intracellular staining of immune cells, and markers of apoptosis in the liver. Results ST2 deficient mice developed significantly more severe hepatitis and had significantly higher number of mononuclear cells in the liver, CD4+ and CD8+ T cells, NKp46+ and CD3+NKp46+ cells, and F4/80+ macrophages. The level of pro-inflammatory cytokines in the sera and number of TNF alpha, IFN gamma, and IL-17 producing cells was higher in ST2 deficient mice. In contrast, number of CD4+Foxp3+ cells was statistically higher in wild type mice. Additionally, treatment of wild type mice with single (1 μg) injection of Interleukin 33 led to attenuation of the liver injury and milder infiltration of mononuclear cells, increase in total number of liver CD4+Foxp3+ cells and IL-4 producing CD4+ T cells. Interleukin 33 also suppressed the activation of caspase 3, prevented the expression of BAX, and enhanced the expression of antiapoptotic Bcl-2 in the liver. Conclusions We concluded that Interleukin 33/ST2 axis downregulated Concanavalin A-induced liver injury and should be evaluated as potential target in fulminant hepatitis in humans.
•Cytokine levels in patients group were heterogeneous, spread in wide range of values.•Grouping data according to disease-relevant factors showed high intragroup diversity.•There was no correlation ...between cytokine levels and each disease-relevant factor.•Inconsistency of literature data is a result of individual differences of patients.
The data addressing cytokine profile in chronically infected HCV patients are conflicting, ranging from Th1 or Th2 cytokine prevalence to the expression of both types of cytokines. Therefore, the aim of this study was to evaluate cytokine profile in these patients. Cytokine sera levels in HCV patients and healthy controls were evaluated using 13plex FlowCytomix Multiplex. Median values of both proinflammatory and anti-inflammatory cytokines were lower in HCV patients then in controls. In addition, the number of subjects producing detectable quantities of cytokines was significantly lower in the group of HCV patients. Yet, cytokine levels in those patients were remarkably heterogeneous ranging from low to extremely high, much higher than the maximal values in control group. Similarly, grouping data according to HCV genotype, HCV RNA load, ALT/AST ratio and the stage of fibrosis showed marked standard deviations, reflecting high intragroup diversity. No correlation was found between each disease-related factor and cytokine levels. Patients investigated in our and similar studies were disparate pursuant to characteristics of the hosts, pathogen and course of the disease. Therefore, the inconsistency of the literature data regarding cytokine pattern in chronic HCV patients may be a consequence of the disregarded/overlooked heterogeneity of these patients.
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