Acridine Orange And Ethidium Bromide Research Articles

In vitro evaluation of curcumin-loaded chitosan-coated hydroxyapatite nanocarriers as a potential system for effective treatment of cancer

Nanotechnology has many potential applications in cancer treatment. For example, nano-drug delivery systems (NDDS) with high bioavailability, biodegradability, and biocompatibility have been developed, in order to increase the therapeutic effects of anticancer drugs. Among these NDDS, high-performance hydroxyapatite (HA) nanoparticles are rapidly advancing in the targeted cancer treatment due to their numerous benefits. Curcumin is an herbal metabolite that acts as a chemical inhibitor through the inhibition of tumor cells and the progression of many cancers. However, the poor bioavailability of curcumin is the most important challenge in using this substance. In this study, HA nanoparticles coated by chitosan were used as a pH-sensitive biopolymer to improve the efficiency and bioavailability of curcumin. For this purpose, HA nanoparticles were first synthesized by the sol-gel method. Then, a layer of chitosan was coated on it, and the curcumin drug was encapsulated in the nanocarrier, under controlled conditions. Techniques such as scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to characterize the nanocarriers. In the second part, nano-drugs prepared by various bioassays were examined. For this purpose, the rate of cytotoxicity by the methyl-thiazol-tetrazolium (MTT) assay and the rate of apoptosis induction by the acridine orange and ethidium bromide (AO/EB) staining method on the brain carcinoma U87MG cell line were investigated.

Lagerstroemia speciosa (L.) Pers. triggers oxidative stress mediated apoptosis via intrinsic mitochondrial pathway in HepG2 cells.

Hepatocellular carcinoma is the second leading cause of cancer-related mortality worldwide. Lagerstroemia speciosa Pers. (Lythraceae) commonly known as Banaba has been used in different forms in traditional medicinal systems for treating various diseases which include diabetes and obesity. In this study, we investigated the cytotoxic potential of ethanolic Banaba leaf extract (EBLE) in HepG2 cells. The phytochemical analysis of EBLE was performed by HPTLC. HepG2 cells were treated with EBLE at 25, 50, 100, and 150 μg/mL concentrations, and cytotoxicity was evaluated by MTT assay. Oxidative stress was assessed by the evaluation of lipid peroxidation, superoxide dismutase, and reduced glutathione. Apoptosis-related morphology was investigated by acridine orange and ethidium bromide (AO/EB) dual staining. Mitochondrial membrane potential (ΔΨm) was evaluated by JC-1 staining. Apoptosis-related marker genes were evaluated by qPCR. HPTLC analysis confirmed the presence of corosolic acid (12.87 μg/mg), berberine (3.19 μg/mg), and gallic acid (2.94 μg/mg) in EBLE. EBLE treatments caused significant and concentration-dependent cytotoxicity and oxidative stress in HepG2 cells. Dual staining with AO/EB confirmed membrane distortion and nuclear chromatin condensation upon EBLE treatments. JC-I staining revealed the loss of ΔΨm. Furthermore, at a molecular level, EBLE treatments interfere with Bax/Bcl-2 homeostasis and induced the pro-apoptotic marker genes such as cytochrome c, Apaf-1, and caspases 9 and 3. EBLE treatments caused cytotoxicity in HepG2 cells, and this could be due to the induction of oxidative stress and apoptosis via the intrinsic or mitochondrial pathway.

Synthesis of SiO2@Cu2-xSe nanospheres for efficient near-infrared radiation mediated treatment and care of gastric cancer patients

Rational design and fabrication of suitable optical absorbing agents (OAAs) are of great significance for state-of-the-art photothermal cancer therapy. Copper selenides have recently emerged as a type of promising photothermal operators thanks to their engineered effortlessness, high carrier concentration, and superior photothermal transformation. Nevertheless, the further in vivo applications are hampered owing to their poor biocompatibility and hydrophobicity. Herein, we synthesized the judicious structure of Cu2-xSe nanospheres coated with mesoporous silica for improving their biocompatibility and hydrophilicity. The Cu2-xSe@mSiO2 core–shell nanospheres were studied by scanning/transmission electron microscopy (SEM/TEM), X-ray (XRD and XPS), and UV–Visible techniques. Such nanocomposite achieved great ability in photothermal removal of cancer cells with little toxicity. The cell death mechanism has been investigated through typical biochemical assays including acridine orange and ethidium bromide (AO-EB), flowcytometry, and nuclear staining. In addition, the cell cycle arrest in human gastric cancer cells (GCCs) has also been evaluated. Strikingly, the in vivo chemo-photothermal therapy of as-prepared Cu2-xSe@mSiO2 toward GCCs confirms the excellent antitumor activity of the core-shell nanospheres under near-infrared radiation (NIR) radiation. It is firmly believed that the Cu2-xSe@mSiO2 nanocomposites hold great potential to function as OAAs for NIR mediated treatment and care of gastric cancer patients.

Antitumor Effects of Chrysophanol in Malignant Optic Nerve Meningioma Cell Lines are Mediated via Caspase Activation, Induction of Mitochondrial Mediated Apoptosis, Mitochondrial Membrane Depolarization and Targeting the Mitogen-Activated Protein Kinase Signaling Pathway

Anthroquinones are considered remarkable anticancer agents. Chrysophanol is an important anthroquinone and it has shown to have the potential to inhibit the growth of the range of cancers. However, there are no studies regarding the anticancer effects of chrysophanol against the malignant meningioma of optic nerve. In this review, the potential of chrysophanol in the treatment of malignant -meningioma of optic nerve was explored by evaluating its anticancer activity against the malignant meningioma CH157-MN cells. The 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay was used for cell viability determination. The 4',6-diamidino-2-phenylindole (DAPI), acridine orange and ethidium bromide (AO/EB) and annexin V/PI assays were used to determine the induction of apoptosis. The potential of reactive oxygen species and the mitochondrial membrane was estimated by flow cytometry. Western blot analysis was performed to determine the protein expression. The results showed that chrysophanol caused significant decline in the viability of the CH157-MN cells and exhibited an IC50 of 30 µmol/L. Anticancer effects were found to be due to the induction of apoptosis as evident form the DAPI and AO/EB staining. The annexin V/PI staining revealed that the apoptotic cells increased from 1.77% in control to 37.21% at 60 µmol/L concentration of chrysophanol. The Bcl-2/Bax expression ratio was decreased and the caspases-3 and 9 were activated upon chrysophanol treatment of the CH157-MN cells. Chrysophanol also triggered the formation of reactive oxygen species and reduction of the mitochondrial membrane potential in the CH157-MN cells and also blocked the Mitogen-activated protein kinase signaling pathway. The findings of the present study suggest that chrysophanol may prove beneficial in the treatment of malignant meningioma of optic nerve. Key Message: The study revealed the anticancer potential of chrysophanol against the malignant optic nerve meningioma.

Isolation, Identification and Characterization of marine bacteria from the deep sea sediment of Bay of Bengal, India, and their antimicrobial and cytotoxicity potential.

A total of 108 marine bacteria were isolated from the deep sea sediment of Bay of Bengal, India. Of which, 15 bacteria showed antimicrobial activity against human pathogenic bacteria. These antagonistic marine bacteria were characterized phenotypically and their taxonomic affiliations were made on the basis of 16S rRNA gene homology and molecular phylogeny tree analysis, the antagonistic marine bacteria were identified as the species of Bacillus, Halobacillus, Staphylococcus, Marinobacter. The extracts of potent marine bacteria exhibited differential cytotoxicity towards lung (A549), cervical (HeLa), breast (MCF-7) and colon (HT-29) cancer cells. The acridine-orange and ethidium bromide (AO/EB) staining of the extract of strain MB30-treated cancer cells showed typical characteristics of apoptosis such as nuclear condensation, cell shrinkage and formation of apoptotic bodies. Present investigation, reports potent marine bacteria from the deep sea sediment of Bay of Bengal that exhibit broad-spectrum antimicrobial and cytotoxicity potential. Due to their innate bioactive potential, these bacteria can be used as the source of potent molecules.

Clerodendrum volubile inhibits key enzymes linked to type 2 diabetes but induces cytotoxicity in human embryonic kidney (HEK293) cells via exacerbated oxidative stress and proinflammation

The toxicity and safety associated with the use of medicinal plants remains a major concern. In this study, the antidiabetic properties of the dichloromethane (DCM) fraction of C. volubile leaves were investigated in vitro. Its cytotoxic effect and mechanism of toxicity were also investigated in Human Embryonic Kidney (HEK293) cells. The fraction was subjected to in vitro antioxidant assays using the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging and Ferric reducing antioxidant power (FRAP) protocols. Its enzyme-inhibitory properties were investigated on α-glucosidase and α-amylase activities. Gas Chromatography Mass Spectroscopy (GCMS) and Fourier Transform Infrared (FTIR) spectroscopic analysis were used to identify its phytoconstituents. Cytotoxicity was determined via MTT assay. The treated cells were assayed for reduced glutathione (GSH), non-protein thiol, nitric oxide and malondialdehyde (MDA) levels, as well as Superoxide Dismutase (SOD), catalase, myeloperoxidase and ATPase activities. Cell apoptosis and/or morphological changes were determined using the acridine orange and ethidium bromide (AO/EB) dual staining method. The fraction showed significant (p < 0.05) antioxidant and enzyme-inhibitory activity. It showed significant (p < 0.05) cytotoxic effect against HEK293 cells with concomitant depletion of antioxidative and elevation of proinflammatory biomarkers. Morphological changes were examined in the cells with an apoptotic index of 0.84. 1,1-Dodecanediol, diacetate was identified as the most predominant compound, while aromatics and amines as the most functional groups present in the fraction. These results suggest the antidiabetic and cytotoxic effects of C. volubile leaves. The toxicity can be attributed to induced oxidative stress and proinflammation with concomitant depletion of ATP leading to apoptosis of the cells.

Genotoxicity and apoptotic activity of biologically synthesized magnesium oxide nanoparticles against human lung cancer A-549 cell line

The study focussed on the synthesis of magnesium oxide (MgO) nanoparticles from an aqueous extract of Penicillium species isolated from soil. A suitable amount of magnesium nitrate (MgNO3) was mixed with the aqueous extract of Penicillium. Then the colour of the solution changed due to the formation of MgO nanoparticles. These nascent formed MgO nanoparticles were further confirmed by using UV spectrophotometry which showed the maximum absorption at 215 nm indicating the formation of MgO nanoparticles. Fourier transform infrared spectroscopy (FTIR) was used to find the possible functional groups and proteins involving the stabilization of MgO nanoparticles. Transmission electron microscopy (TEM) study revealed the size, the shape as well as the dispersity of the prepared MgO nanoparticles and showed that they were well dispersed around 12–24 nm (scale 200 nm). The anticancer activity against A-549 cell line of these green synthesized MgO nanoparticles was evaluated. The result showed good anticancer effect after 24 h of incubation. Nevertheless these MgO nanoparticles showed less effect on normal Vero cells. Further apoptotic study clearly displayed the effect of MgO nanoparticles on cancer cells. The effect was observed through chromatin condensation by forming apoptotic bodies using propidium iodide, acridine orange and ethidium bromide (AO/EB) staining technique. The DNA was isolated to confirm the DNA damage; the observation clearly showed DNA damage when compared with DNA ladder.

In Vitro Apoptosis Induction in a Human Prostate Cancer Cell Line by Thermotolerant Glycolipid from Bacillus licheniformis SV1

AbstractA highly potent glycolipid was isolated from a crude oil‐contaminated soil bacterium Bacillus licheniformis SV1 (NCBI GenBank Acc. No. KX130852) when grown on a modified mineral salt medium supplemented with 2% oleic acid. The maximum reduction in surface tension of cell‐free broth from 71 ± 0.812 to 25.919 ± 0.984 mN/m with 89 ± 0.346% emulsification activity was recorded after 120 h of growth. Glycolipid was purified using chromatographic techniques and the presence of aliphatic chain (C–H stretch) and OH‐band was revealed by NMR, GC–MS and FTIR analysis. Stability of glycolipid up to 250 °C and its complete decomposition at 507 °C was recorded by thermogravimetric analysis (TGA). MTT assay (IC50 = 0.473 ± 0.048 mg/ml) along with 4′,6‐diamidino‐2‐phenylindole (DAPI) and acridine orange and ethidium bromide (AO/EtBr) staining validated that glycolipid induces cell apoptosis in human prostate cancer cell line (PC‐3). Furthermore, potent anti‐cancerous compounds such as 9‐octadecanoic acid (22.55%), linoleic acid methyl ester (2.2%) and palmitic acid (1.18%) were also detected in the GC–MS spectra of the purified glycolipid.

Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant–DSBs versus alkaline–DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

Vitamin E pretreatment prevents the immunotoxicity of dithiocarbamate pesticide mancozeb in vitro: A comparative age-related assessment in mice and chick

Pesticides used for crop protection cause life-threatening diseases affecting the immune system of non-target organisms including birds and mammals. Functionality of immune system is age-dependent; early- as well as old-life stages are more susceptible to toxic exposures because of less competent immune system. Vitamins are so far known to reduce toxic effect of several pesticides and/or xenobiotics. The present in vitro study elucidated immunotoxicity of fungicide mancozeb through comparable stages of immune system maturation in mice (1, 3, and 12months) and chicks (4, 8, and 11weeks). In vitro splenocytes viability on exposure to mancozeb was quantitatively assessed by MTT assay and qualitatively by acridine orange and ethidium bromide (AO/EB) double fluorescence staining. Mancozeb exposure dose dependently (250, 500, 1000, 2500, 5000 and 10,000ng/ml) decreased the splenocytes viability. The in vitro preventive effect of Vitamin E has also been explored on toxicity induced by mancozeb. The increased susceptibility observed both in early and aged groups was due to less/decline competence of the immune system.

One-pot synthesis of podophyllotoxin-thiourea congeners by employing NH₂SO₃H/NaI: Anticancer activity, DNA topoisomerase-II inhibition, and apoptosis inducing agents.

A facile one-pot method for the synthesis of novel podophyllotoxin-thiourea congeners has been developed by using NH2SO3H/NaI system. Interestingly, 4β-azido podophyllotoxin reduction with concomitant aryl isothiocyanates coupling under mild reaction conditions has been achieved. These compounds have been investigated for their in vitro cytotoxicity against A549, MDA MB-231, DU-145, LNCaP, and HGC-27 cancer cell lines. Some of the representative compounds have selectively exhibited cytotoxicity on DU-145 (human prostate cancer) cells and the most potent compound was 4a (IC50 of 0.50 ± 0.03 μM) with optimal safety therapeutic window (81.7 fold) on normal human prostate cell line (RWPE-1, IC50 of 40.85 ± 0.78). The flow-cytometric analysis of the compound 4a in prostate cancer cells indicated a strong G2/M-phase arrest and significant topoisomerase II inhibition activity. Furthermore, these compounds induce apoptosis as observed by Acridine Orange and Ethidium Bromide (AO/EB) staining and Annexin V binding assay. Molecular docking results of the title compounds with topoisomerase-IIα were presented as theoretical support for the experimental data.

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.

Cytotoxicity of zinc oxide nanoparticles and silver nanoparticles in human epithelial colorectal adenocarcinoma cells

Safety concerns related to nanoparticles have stimulated research in this area. Therefore, the objective of this study was to compare the cytotoxicity of ZnO NPs with Ag NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells. Nanoparticles were firstly characterized by size in transmission electron microscope. The WST-1 assay displayed cells exposed to higher nanoparticles concentrations and for longer time showed more serious cytotoxic changes. The acridine orange and ethidium bromide (AO/EB) double staining characterized the morphology of viable cells, apoptotic cells and necrotic cells. Both ZnO and Ag NPs cytotoxicity exhibited a significant depletion of superoxide dismutase (SOD) level with an increase in reactive oxygen species (ROS) level. Our conclusion suggested that ZnO NPs may exert higher cytotoxicity than Ag NPs in the same concentrations range. For ZnO NPs, cytotoxicity exhibited a dose- and time-dependent effects. In contrast with low concentrations Ag NPs, cells showed the subsequence of hormesis. There is still little knowledge about how nanoparticles in food packing materials interact with the cell membrane, and the consequence of these interactions.

Acalypha indica Linn: Biogenic synthesis of silver and gold nanoparticles and their cytotoxic effects against MDA-MB-231, human breast cancer cells

This study reports the in vitro cytotoxic effect of biologically synthesized silver and gold nanoparticles against MDA-MB-231, human breast cancer cells. Formation of silver and gold nanoparticles was observed within 30min and the various characterization techniques such as UV–vis spectrophotometer, FE-SEM, TEM and XRD studies were confirmed the synthesis of nanoparticles. Further, MTT, acridine orange and ethidium bromide (AO/EB) dual staining, caspase-3 and DNA fragmentation assays were carried out using various concentrations of silver and gold nanoparticles ranging from 1 to 100μg/ml. At 100μg/ml concentration, the plant extract derived nanoparticles exhibited significant cytotoxic effects and the apoptotic features were confirmed through caspase-3 activation and DNA fragmentation assays. Thus, the results of the present study indicate that biologically synthesized silver and gold nanoparticles might be used to treat breast cancer; however, it necessitates clinical studies to ascertain their potential as anticancer agents.

Excretion Product of Shigella dysenteriae (SdyEP) Induced Cell Death in Early Larval Stage of Zebrafish (Danio rerio): Acridine Orange and Ethidium Bromide (AO/EB) in vivo Staining

Excretion Product of Shigella dysenteriae (SdyEP) Induced Cell Death in Early Larval Stage of Zebrafish (Danio rerio): Acridine Orange and Ethidium Bromide (AO/EB) in vivo Staining

Excretion Product of Shigella dysenteriae (SdyEP) Induced Cell Death in Early Larval Stage of Zebrafish (Danio rerio): Acridine Orange and Ethidium Bromide (AO/EB) in vivo Staining

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de celulas, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excrecion de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recoleccion de los huevos y larvas de pez cebra, asi como la obtencion del producto de la excrecion, se realizaron de acuerdo a los estandares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del PESdy, se expreso como dependiente de la concentracion y del tiempo de exposicion. La poblacion de larvas sobrevivientes, presentaron curvatura troncal en un 10%, en relacion a los controles. La necrosis se puso en evidencia a traves de la opacidad de las larvas despues de 24 h. El uso de NA/BE revelo un patron de distribucion de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una gran poblacion de celulas muertas, alrededor de la region anal y caudal, se ponen en evidencia por la presencia de nucleos de naranja en mayor numero que en los controles. Los resultados apoyan la aplicacion de la coloracion NA/BE en modelo experimental de pez cebra, para la evaluacion de la accion toxica de nuevas moleculas y nuevos productos de excrecion bacterial con potencial terapeutico.

Kinetin induces cell death in root cortex cells of Vicia faba ssp. minor seedlings

The double fluorescence staining with acridine orange and ethidium bromide (AO/EB) revealed that treatment of Vicia faba ssp. minor seedlings with kinetin-induced programmed cell death (PCD) in root cortex cells. Kinetin-induced cell death reflected by the morphological changes of nuclei including their invagination, volume increase, chromatin condensation and degradation as well as formation of micronuclei showed by AO/EB and 4,6-diamidino-2-phenylindol staining was accompanied by changes including increase in conductivity of cell electrolytes secreted to culture media, decrease in the number of the G1- and G2-phase cells and appearance of fraction of hypoploid cells as the effect of DNA degradation without ladder formation. Decrease in the number of mitochondria and in the activity of cellular dehydrogenases, production of reactive oxygen species (ROS), appearance of small and then large lytic vacuoles and increase in the amount of cytosolic calcium ions were also observed. The PCD was also manifested by increased width and weight of apical fragments of roots as well as decreased length of cortex cells which led to shortening of the whole roots. The kinetin-induced PCD process was almost completely inhibited by adenine, an inhibitor of phosphoribosyl transferase, and mannitol, an inhibitor of ROS production. These cell-death hallmarks and pathway of this process suggested that the induction of kinetin-specific vacuolar type of death, expressed itself with similar intensity on both morphological and metabolic levels, was a transient protecting whole roots and whole seedlings against elimination.

Novel selective estrogen receptor modulators induce LNCaP cell death via mechanisms involving apoptosis

Selective Estrogen Modulators (SERMs) offer an alternative approach for treating prostate cancer that differs from the treatment options that are currently available. Using the LNCaP cell line we evaluated three novel SERMs, 3‐[2‐diethylamino]‐ethoxy]‐]‐D 1,3,5(10)‐estrien‐17‐one (DE), 3‐[2‐diisopropylamino]‐ethoxy‐ D1,3,5(10)‐estrien‐17‐one (DI) and 17‐b‐3‐[2‐piperidinyl]‐ethoxy]‐ ]‐D 1,3,5(10)‐estrien‐17‐ol (EPOH), which are aminoalkyloxy derivatives of 1,3,5(10)‐Estratrien. The cytotoxic effect (judged by cell viability) of DE, DI, EPOH and 4‐hydroxytamoxifen (4‐OHT) was determined using the Alamar Blue assay. The effect of the SERMs on apoptosis was determined by Acridine Orange and Ethidium Bromide (AO/EB) staining method. It was observed in comparison to the control, that the three experimental compounds caused a dose‐dependent decrease in cell viability (EC50 values ranging between 300 μM – 400 μM). AO/EB staining indicated the induction of apoptosis at the same concentrations that caused the dose‐dependent increase in cell death by DE, DI and EPOH. Taken together these results indicate that the novel SERMs cause cell death via underlying mechanisms that lead to apoptosis/programmed cell death and with further evaluation will reveal the potential of these novel SERMs for the treatment of prostate cancer. Supported by NIH/NCRR/RCMI Grant (G12 RR0 3020).

Earthworm fibrinolytic enzyme: anti-tumor activity on human hepatoma cells in vitro and in vivo

The earthworm fibrinolytic enzyme (EFE) is a complex protein enzyme that is widely distributed in the earthworm's digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direct effect on fibrin, but also can activate plasminogen. Its therapeutic and preventative effects on thrombosis-related disease have been confirmed clinically. Recently, there has been increased interest in the anti-tumor activity of EFE. In this study, the anti-tumor activity of EFE, isolated from Eisenia foetida, on human hepatoma cells was evaluated in vitro and in vivo. The potential mechanisms involved were also studied. In vitro experiments were performed in four human hepatoma cell lines: HLE, Huh7, PLC/PRF/5 and HepG2. After treatment with EFE in various concentrations, the inhibition of the rate of cell proliferation was measured. For the in vivo studies, tumor-bearing models xenografted with Huh7 cells were developed in nude mice, and then the mice were fed with EFE once a day for 4 weeks, and the control group received only saline. An inhibitory effect on tumor growth was observed. Also, apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange and ethidium bromide (AO/EB). The expression of matrix metalloproteinase 2 (MMP-2) were detected by Western blotting assay. After treatment with various concentrations of EFE, the proliferation of all hepatoma cell lines was suppressed to varying degrees in vitro. The IC(50) for HLE, Huh7, PLC/PCF/5 and HepG2 were 2.11, 5.87, 25.29 and 17.30 uku/ml, respectively. After administration of EFE orally for 4 weeks, the growth of tumor xenograft of Huh7 cells in nude mice was significantly inhibited in vivo. The tumor inhibitory rates in the EFE 500 uku/(kgxd) and 1000 uku/(kgxd) groups were 46.08% (compared with control group, P = 0.026) and 57.52% (compared with control group, P = 0.002) respectively. Meanwhile, the average weight of body, spleen or thymus did not show any remarkable differences among the various groups. The population in sub-G(1) stage was more in the EFE treated groups than in the control group according to flow cytometric assay. After treatment with EFE 0, 5, 10 uku/ml for 72 hours, the apoptotic rates were 3.5%, 10.9% and 12.3% in HLE cells, and 5.0%, 24.7% and 34.5% in Huh7 cells respectively. Under fluorescent staining with AO/EB, the apoptotic morphological changes could be detected more significantly in the EFE treated groups than in the untreated groups. After treatment with EFE in doses of 0, 5, 10 uku/ml for 72 hours, the apoptotic rates were 3.02%, 8.76%, 10.54% in HLE cells, and 3.95%, 18.27%, 30.89% in Huh7 cells respectively. The apoptosis-inducing effects of EFE occurred in a dose dependent manner. Western blotting assay showed that, after treatment with EFE, the secretions of MMP-2 were significantly inhibited in HLE and Huh7 cells. EFE showed significant anti-tumor activity in hepatoma cells both in vitro and in vivo, which may be because EFE could induce apoptosis of hepatoma cells and inhibit the expression of MMP-2. This suggests that EFE has a potential role in the treatment of hepatoma.