Journal: EMBO Reports

Article Title: New androgen receptor genomic targets show an interaction with the ETS1 transcription factor

doi: 10.1038/sj.embor.7401046

Figure Lengend Snippet: Androgen receptor-bound gene promoters identified by ChIP-chip. ( A ) AR ChIP-chip profiles of eight AR target gene promoters from vehicle (+EtOH)- and androgen (+R1881)-treated experiments. Genomic locations are indicated on the x axis and enrichment compared with total input DNA is shown on the y axis. ( B ) AR ChIP and qPCR analysis of 26 AR promoter targets in LNCaP cells. ( C ) AR ChIP and qPCR analysis of eight promoter targets in DUCaP cells. Relative enrichment by ChIP was quantified by using the standard curve method following 1 h with vehicle (EtOH) or androgen (R1881) relative to β-actin control and total genomic DNA input (see the online for details). Values are the averages of replicate experiments. Enrichment scores (S) and Wilcoxon scores (W) from LNCaP AR ChIP-chip analysis are indicated next to corresponding genes. AR, androgen receptor; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with on-array detection; Chr, chromosome; EtOH, ethyl alcohol; KLK, human glandular kellikrein; qPCR, quantitative PCR.

Article Snippet: LNCaP cells lysates were incubated with AR (N20, Santa Cruz), ETS1 (C20, Santa Cruz) or control rabbit IgG antibodies.

Techniques: ChIP-chip, Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: EMBO Reports

Article Title: New androgen receptor genomic targets show an interaction with the ETS1 transcription factor

doi: 10.1038/sj.embor.7401046

Figure Lengend Snippet: ETS1 and androgen receptor interaction. ( A ) Sequence logos for ETS1 and AR ChIP-chip-enriched motifs 9 and 1. ( B ) UCSC Genome Browser tracks showing 15-bp ARE, 6-bp AR ‘half-site' and ETS1 motif occurrence in UNQ9419, CCNG2 and PRAME promoters. Locations of AR ChIP-chip peaks are indicated; darker boxes represent higher signal. ( C ) ETS1 ChIP and qPCR for UNQ9419, CCNG2 and PRAME promoters from vehicle (+EtOH)- and androgen (R1881)-treated experiments. ( D , E ) Quantitative real-time PCR of ETS1, UNQ9419, CCNG2 and PRAME 48 h after transfection with an ETS1 RNAi construct (pSR-ETS1) or a scrambled control (pS-Scr) ( D ) and ETS1 expression construct (+ETS1) or an empty vector control (pSG5) ( E ). qPCR data were quantified using the standard curve method, values relative to control sample and averages of replicate experiments. ( F , G ) Confocal microscopy images of LNCaP cells treated with androgen (+R1881) or vehicle (+EtOH) for 1 h. Cells were transfected with either AR–GFP expression construct ( F ) or pSG5 empty vector ( G ) and stained for AR (N20) and ETS1 (C20 and 1G11), as indicated. AR, androgen receptor; ARE, androgen-receptor elements; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with on-assay detection; DAPI, 4′, 6′-diamidino-2-phenylindole; ETS, avian erythroblastosis virus E26 homologue; GFP, green fluorescent protein; qPCR, quantitative PCR; RNAi, RNA interference.

Article Snippet: LNCaP cells lysates were incubated with AR (N20, Santa Cruz), ETS1 (C20, Santa Cruz) or control rabbit IgG antibodies.

Techniques: Sequencing, ChIP-chip, Real-time Polymerase Chain Reaction, Transfection, Construct, Control, Expressing, Plasmid Preparation, Confocal Microscopy, Staining, Chromatin Immunoprecipitation, Virus

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Cytotoxic effect of Buxus natalensis leaf extracts on HepG2 ( A ), LNCaP ( B ), and Chang liver ( C ). Ten thousand cells were seeded per well for each cell line on 96-well microtiter plates, and the cells were treated for 48 h with different leaf extracts (15.625–500 µg/mL) under standard cell-culture conditions. The cell viability was estimated as a percentage of cells treated with DMSO (0.5%) considered as 100%. B. natalensis hydroethanolic leaf extract (BNHLE); B. natalensis methanolic extract (BNMLE); B. natalensis aqueous leaf extract (BNALE).

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Colony formation after treatment with B. natalensis hydroethanolic leaf extract (BNHLE). One thousand cells from each cell line HepG2 ( A ), LNCaP ( B ), and Chang liver ( C ) were seeded in 6-well microtiter plates, and exposed to BNHLE for 24 h, followed by the replacement of the culture medium every 3 days for 15 days. The colonies were stained with crystal violet (0.5%) and allowed to dry overnight. Then, 2 mL of acetic acid (33%) was added per well, and the absorbance was read at 550 nm using a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA). One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Intracellular ROS production in LNCaP cells after treatment with B. natalensis hydroethanolic leaf extract (BNHLE). LNCaP cells (10,000 cells/well) were treated for 45 min with 100 µL of 20 µM of DCFH-DA diluted in culture medium containing 2% FBS. Thereafter, these cells were exposed for 24 h to BNHLE (100, and 200 µg/mL), and hydrogen peroxide (H 2 O 2 , 50 µM) or DMSO (0.5%) used as negative control (Ctrl). Images were captured using a Flexacam C1 camera (Leica Microsystems GmbH, Wetzlar, Germany) connected to a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) using an excitation/emission filter (480/535 nm) on 20× objective ( A ). The cell fluorescence was measured at the wavelengths of 485 nm (excitation) and 535 nm (emission) on a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA), and intracellular ROS levels were expressed as percentage of negative control cells ( B ). One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Negative Control, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Caspase luminescence activity in HepG2 ( A , B ) and LNCaP cells ( C , D ) after exposure to B. natalensis hydroethanolic leaf extract (BNHLE). HepG2 and LNCaP cells (10,000 cells/well) were exposed for 18 h to BNHLE and doxorubicin hydrochloride (Doxo, 2 µM) or DMSO (0.5%) used as negative control (Ctrl). Caspase reagents were added in equal volume as the culture medium, and luminescence was read using SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA). Caspase activities were expressed as a percentage (fold change) of cells treated with DMSO (0.5%) used as a negative control. One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically significantly different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Activity Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Expression levels of human p53 and NF-κB-p65 in HepG2 ( A , B ) and LNCaP ( C , D ) after 24 h of treatment with B. natalensis hydroethanolic leaf extract (BNHLE). Cells were treated for 24 h with BNHLE, or DMSO (0.5%) used as negative control (Ctrl). Equal amounts of proteins in cell lysates were used for the quantification of the expression levels of NF-κB-p65 and p53 using commercially available ELISA kits (Cat# E-EL-H1388 and Cat# E-EL-H0910, Elabscience Biotechnology Inc., Houston, TX, USA). One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. # means statistically significantly different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Expressing, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Expression levels of human BAX and BCL-2 in LNCaP ( A , B ) and HepG2 ( C , D ) as well as their respective BAX/BCL-2 ratio ( E , F ) after 24 h of treatment with B. natalensis hydroethanolic leaf extract (BNHLE). Cells were treated for 24 h with BNHLE, or doxorubicin hydrochloride (Doxo, 2 µM) or DMSO (0.5%) used as negative control (Ctrl). Equal amounts of proteins in cell lysates were used for the quantification of the expression levels of BAX and BCL-2 using commercially available BCL-2 Associated X protein and BCL-2 ELISA kits (Cat# EEL030 and Cat# BMS244-3, Thermo Fisher Scientific, Massachusetts, MA, USA), respectively. The BAX/BCL-2 ratio was calculated by dividing the BAX expression level against the BCL-2 level at each tested concentration. One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically significantly different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Concentration Assay