Antioxidant and Anti-Cancer Potentials of Rheum emodi Rhizome Extracts

The objective of this study was to determine antioxidant and cytotoxic efficacies of methanolic and aqueous extracts of Rheum emodi Wall. ex Meissn. rhizome. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activities, inhibitory effect on lipid peroxidation and Fe3+ reducing antioxidant property have been used to investigate antioxidant properties of the extracts. Cytotoxicity of the extracts was tested on MDA-MB-435S and Hep3B cell lines. Both extracts displayed extensive cytotoxicity to the tested cell lines. The extracts were studied for their ability to protect pBR322 DNA from damage by UV induced photolysis of H2O2. The aqueous extract, though inferior to methanolic extract in its antioxidant potential exhibited efficiency in DNA protection, while the methanolic extract failed to protect the DNA. The amount of total polyphenolics in the extracts was measured by spectrophotometric method. The methanolic extract contained higher polyphenolic contents than aqueous extract. Significant positive correlations were observed (P < .05) between results of phenolic content estimation and that of antioxidant assays. Hence, high-performance liquid chromatography analysis was performed to identify few major phenolic compounds that might be responsible for these therapeutic properties. These results indicate that rhizome of R. emodi possesses antioxidant and cytotoxic activities and therefore have therapeutic potential

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism

Digitoxin Analogues with Improved Anticytomegalovirus Activity

Cardiac glycosides are potent inhibitors of cancer cell growth and possess antiviral activities at nanomolar concentrations. In this study we evaluated the anticytomegalovirus (CMV) activity of digitoxin and several of its analogues. We show that sugar type and sugar length attached to the steroid core structure affects its anticytomegalovirus activity. Structure–activity relationship (SAR) studies identified the l-sugar containing cardiac glycosides as having improved anti-CMV activity and may lead to better understanding of how these compounds inhibit CMV replication

Emetine achieves high tissue concentrations and is efficacious against MCMV replication.

<p><b>A)</b><i>In vivo</i> pharmacokinetics of emetine. Plasma, liver, lung and spleen samples were collected at the indicated time points after single oral administration of emetine at 0.1 mg/kg in male BALB/c mice. Concentrations were measured using UPLC-MS/MS methods. For plasma, the concentrations observed were mostly below the level limit of quantification. <b>B)</b> Quantitative real-time PCR of viral gB was performed on DNA extracted from blood at day 14 post infection. Plaque assay was performed from salivary glands <b>C)</b> or liver <b>D)</b> collected at day 14 post infection. Emetine was administered orally starting 24 hpi or 72 hpi at 0.1 or 1.0 mg/kg every 3 days. GCV dose was 10 mg/kg/dose administered intraperitoneally twice daily.</p

Nuclear localization of RPS14 persists during infection and emetine treatment in low-density cells.

<p><b>A)</b> Cells were seeded at 0.5 million/4-well chamber slide, infected and treated with either emetine (75 nM) or GCV (5μM) for 24 h. Cells were stained with IE1/2 (Alexa 555:Red) for evidence of infection and RPS14 (FITC: Green) and nuclear DAPI. Stained slides were subjected to confocal microscopy and nuclear localization was quantified using NIS elements software (Nikon). <b>B)</b> The same procedure was repeated at 72 hpi. The images shown are a representative of two independent experiments. Percent nuclear localization is represented as mean ± SD of at least 25 cells per condition from two different fields in the slide.</p

The anti-HCMV activity of emetine is reduced in low-density HFFs.

<p><b>A)</b> Cells were seeded at indicated densities in a 96-well plate, infected with pp28-luciferase HCMV Towne (MOI = 1) and treated with emetine (75 nM) or GCV (5 μM). Luciferase activity was measured at 72 hpi and normalized to the activity of infected untreated cells. Data represent mean ± SE of triplicate determinations from a representative of two independent experiments. <b>B)</b> Same cell lysates as in A were collected for Western blotting at 72 hpi and expression of viral proteins UL44 and pp65 was determined. <b>C)</b> Supernatants from each well as in A were used to infect cells (1 million cells in a 96-well plate) in the corresponding well and luciferase activity was measured at 72 hpi. <b>D)</b> Cells were seeded into 12-well plates at 0.5 and 2 million cells/plate, infected with 100 PFU/well of HCMV Towne or MCMV, and treated with emetine (75 nM) or GCV (5 μM). After 10 days (for HCMV) or 3 days (for MCMV) plaques were stained with crystal violet and the number of plaques enumerated. Data shown are average of 2 wells (±SD) for a representative experiment from two different experiments.</p

Emetine disrupts HCMV mediated MDM2-p53 complex.

<p><b>A)</b> MDM2 and p53 expression increases with emetine treatment. Cells were seeded at 0.5 or 2 million cells/plate, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5 μM). Cell lysates were harvested at 24 or 72 hpi for Western blotting. <b>B)</b> Cells were seeded at 2 million cells/plate, infected with HCMV Towne and treated with emetine (75 nM) or GCV (5 μM). RNA was harvested and qRT-PCR was performed for p21. Data represent mean ±SE of triplicate determinations from a representative of three independent experiments <b>C)</b> HFFs were seeded at 1 or 2 million cells/plate and treated with emetine (75 nM) or GCV (5 μM). Cell lysates were harvested at 72 hpi for Western blotting. <b>D)</b> HFFs were seeded at either 0.5 or 2 million/plate in 100 mm dishes, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5μM) for 24h. MG132 (10 μM) was added after 12 h. At 24 hpi, lysates were collected and subjected to IP with anti-MDM2 antibody followed by immunoblotting with anti-p53 antibody or <b>E)</b> IP with anti-p53 antibody followed by immunoblotting with anti-MDM2 antibody. <b>F</b>) A model depicting the mechanism by which emetine disrupts HCMV mediated MDM2-p53 interaction in high-density cells, but not in cycling low-density cells.</p

Emetine is an early inhibitor of HCMV replication.

<p><b>A)</b> Emetine does not inhibit HCMV entry. Cells were treated with emetine (75 nM), GCV (10 μM), and CpG 2006 (10 μM) 24 h prior to infection. Cells were infected with HCMV and treated with compounds for 90 min. Immunofluorescence staining was performed with mouse monoclonal anti-pp65 antibody. The fluorescence of rhodamine anti-mouse IgG and DAPI was visualized and merged using a Nikon Eclipse E-800 fluorescence microscope. <b>B)</b> Emetine has an early activity against HCMV. Cells were infected with HCMV Towne, and compounds were added at 0, 6, 12, 24, 36, and 48 hpi (Add on). Culture supernatants (10%) were collected at 72 hpi for a plaque assay after 14 days. <b>C)</b> Cells were infected with HCMV Towne and treated with compounds immediately after virus adsorption. Compounds were removed at 0, 6, 12, 24, 36, and 48 hpi (Removal). Culture supernatants were collected at 72 hpi for titration by plaque assay. Data represent mean ± SE of triplicate determinations from a representative of two independent experiments.</p