Milan Zaric

Chelidonium majus crude extract inhibits migration and induces cell cycle arrest and apoptosis in tumor cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE Chelidonium majus L (Papaveraceae) is widely used in alternative medicine for treatment of various disorders. Antitumor activities of alkaloids isolated from this plant have been reviewed, while there are only a few studies that examine properties of the whole extract. AIM OF THE STUDY The aim of the present study was to investigate direct cytotoxic effects, as well as indirect antitumor effects of Chelidonium majus ethanolic extract against different tumor cell lines,. MATERIALS AND METHODS MTT and SRB assays were performed to estimate cytotoxic effects of Chelidonium majus extract against human tumor cell lines A549, H460, HCT 116, SW480, MDA-MB 231 and MCF-7 and peripheral blood mononuclear cells from healthy individuals. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by extract was determined by flow cytometry and cell morphology assessment. Inhibitory effect on migration of cancer cells was assessed by wound healing assay. RESULTS Chelidonium majus extract showed selective time- and dose-dependent increase of cytotoxicity in all six cell lines, with individual cell line sensitivities. Extract promoted cell cycle arrest and induced apoptosis. Cotreatment with doxorubicin enhanced cytotoxicity of the drug. Also, inhibitory effect on migration was shown with non-toxic extract concentration. CONCLUSIONS These results indicate possible usefulness of Chelidonium majus crude extract in antitumor therapy, whether through its direct cytotoxic effect, by prevention of metastasis, or as adjuvant therapy.

Chrysin induces apoptosis in peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia.

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.

Naphthoquinone rich Onosma visianii Clem (Boraginaceae) root extracts induce apoptosis and cell cycle arrest in HCT-116 and MDA-MB-231 cancer cell lines

Abstract In the present study, five root extracts of Onosma visianii Clem were investigated for their in vitro cytotoxic activity. On the basis of HPLC-PDA analysis, these extracts have proved to be a rich source of naphthoquinones as natural colourants for food and cosmetic industry. All investigated root extracts contain acetylshikonin, isobutyrylshikonin and α-methylbutyrylshikonin as major compounds. As the most abundant source of active compounds for antitumour therapy, acetone, chloroform and ethyl acetate extracts showed strong cytotoxic activity towards HCT-116 and MDA-MB-231 cancer cell lines. Also, these extracts induced apoptosis and cell cycle arrest in HCT-116 and MDA-MB-231 cancer cell lines.

Crystal and molecular structure of a new palladium(II) complex with a coumarin-valine derivate

The new coumarine derivate with methyl ester of 2-((Z)-1(2,4-dioxochroman-3-ylidene)ethylamino)-3-methylbutanoic acid and the corresponding palladium(II) complex are synthesized and characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy. The proposed structure of the ligand was confirmed based on the X-ray structural study.

Cytokine profile in chronic hepatitis C: An observation

HighlightsCytokine 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. Abstract 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.

Stereospecific ligands and their complexes. XXIV. Synthesis, characterization and some biological properties of Pd(II) and Pt(II) complexes with R2-S,S-eddtyr

Four new platinum(II) complexes of general formula [PtCl2(R2-S,S-eddtyr)] (R = ethyl, n-propyl, n-butyl and n-pentyl); S,S-eddtyr = ethylenediamine-N,N′-di-(2,2′-di(4-hydroxy)-benzyl-acetic acid) have been synthesized and characterized by microanalysis, and infrared, 1H NMR and 13C NMR spectroscopy. The in vitro antimicrobial activity of ligands L1–L4 [L = R2-S,S-eddtyr; R = ethyl (L1), n-propyl (L2), n-butyl (L3), n-pentyl (L4)], platinum(II) complexes C1–C4 [PtCl2(R2-S,S-eddtyr)] [R = ethyl (C1), n-propyl (C2), n-butyl (C3), n-pentyl (C4)] and palladium(II) complexes C5–C8 [PdCl2(R2-S,S-eddtyr)] [R = ethyl (C5), n-propyl (C6) or n-butyl (C7) or n-pentyl (C8)] was investigated. The cytotoxicity of ligands and platinum(II) complexes was investigated using MTT assay. The interaction of platinum and palladium complexes [MCl2(R2-S,S-eddtyr)] (M = Pt or Pd) with calf thymus DNA (CT-DNA) was investigated using UV-Vis absorption and fluorescence spectroscopy. The association constant (Kb) estimated from the absorption spectral study and the quenching constant (KSV) calculated from relevant fluorescence quenching data indicate a non-covalent interaction between the metal complex and DNA. Ethidium bromide (EB) competitive studies revealed that complexes C1–C8 could interact with CT-DNA through intercalation. Furthermore, the interactions between human serum albumin (HSA) and the platinum(II) and palladium(II) complexes were also investigated by UV-Vis absorption and fluorescence spectroscopy, showing that the new complexes could strongly bind with HSA.

Induction of mitochondrial apoptotic pathway by raloxifene and estrogen in human endometrial stromal ThESC cell line

Introduction 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. Material and methods 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. Results 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. Conclusions 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.

Older Hypertensive Patients’ Adherence to Healthy Lifestyle Behaviors

Abstract Non-pharmacological treatment including diet, body weight reduction, smoking cessation and physical activity, is very important part of hypertension treatment. The objective of this study was to investigate the adherence to healthy lifestyle behavior in the representative sample of the older hypertensive patients, and to investigate factors associated with adherence in the studied older population. The study was conducted on random sample of 362 long term hypertensive (> five years) patients older than 65 years of age, at Health Care Center of Kragujevac. Adherence was assessed using the structured questionnaire for the analysis of the implementation of both hypertension and diabetes guidelines in the primary care. Both bivariate and multivariate analyses were conducted. Nearly 35% of examined patients were highly adherent; they exercised regularly, avoided smoking for at least five years and consumed special healthy diet prescribed for hypertension. Another 35.6% of the cases reported exercising regularly, 39.5% followed the recommended diet for the hypertension, while 23.4% of the patients have still consumed cigarettes. Multivariate logistic regression demonstrated that received counseling on healthy lifestyle behaviors by physicians and lack of education predicted high adherence to healthy lifestyle behavior. In order to improve adherence of elderly hypertensive patients to healthy lifestyle, strengthening patient-physician relationships through efforts to enhance communication may be a promising strategy to enhance patients’ engagement in healthy lifestyle behaviors for hypertension. Such an improvement could be achieved through the education of both the physicians and patients.

Enhanced cytotoxicity and apoptosis by raloxifene in combination with estrogen and methotrexate in human endometrial stromal cells

Endometrial hyperplasia is a condition that may lead to the development of endometrial carcinoma. Initially, changes of the endometrium are caused by the estrogens hyperstimulation that may lead to the development of an irregular bleeding and the infertility problems. Therapy of endometrial hyperplasia is limited to medical and surgical approaches. During the past decade, the new types of drugs were developed for the treatment of the endometrial hyperplasia. Here, for the first time, we investigated the cytotoxic effects of the various combinations of estrogen, raloxifene, and methotrexate in human ThESC cell line as a possible potential treatment of the endometrial hyperplasia. Our aim was to investigate and to determine the most efficient combination of investigated drugs in ThESC cells during 24‐hr period using MTT assay, FACS analysis, and immunofluorescence staining. Our results demonstrated that the combination of raloxifene with methotrexate efficiently induced both the cytotoxicity and apoptosis in ThESC cells when compared to their single effect, as well as to the effect of combined treatment of raloxifene with estrogen. The application of the low doses of methotrexate combined with raloxifene offers all advantages of a potential beneficial antitumor match in cancer chemoprevention and therapy.

Enhancement Of Dermal Fibroblast Isolation Method

ABSTRACT Cultivated fibroblasts have been widely used in a large number of in vitro studies. Although they readily proliferate under cell culture conditions, improvements in methods for their isolation are necessary. Here, we present our modified enzyme digestion method and compare its efficiency with commonly used techniques. Three foreskin samples from young, middle-aged and old donors were used. The classical explant, standard enzyme digestion method with collagenase and our improved enzyme digestion method were compared for efficiency of fibroblast isolation and the time needed to achieve 95% confluence in a 30-mm Petri dish. The explant method was the slowest to achieve fibroblast confluence, especially with the tissues from the older donors (up to 23 days). With the standard enzyme digestion method, the skin tissue was partially digested, but the fibroblasts reached confluence much faster (the younger donor cells needed approximately 7 days to reach confluence). Our modified “mixed” enzyme digestion method was the fastest (the fibroblasts from the younger donors needed up to 5 days to reach confluence). For studies requiring the primary isolation and cultivation of dermal fibroblasts, the best method to achieve this goal is the tissue digestion method with the multiple enzyme solution.