Journal:

Article Title: Aminoflavone induces oxidative DNA damage and reactive oxidative species-mediated apoptosis in breast cancer cells

doi: 10.1002/ijc.23244

Figure Lengend Snippet: AF induces cell death in MDA-MB-468 cells. (a) Cells were treated with AF for 12 hr and visualized by Hoffman modulation phase contrast microscopy (×40 magnification). Data is representative of at least 3 independent experiments. (b) MDA-MB-468 cells treated with DMSO (control) and indicated AF concentrations for 24 hr were exposed to Annexin-V-FITC and propidium iodide staining prior to FACS-can analysis. Data represent the mean of at least 3 independent experiments; bars, SEM. **p < 0.01 or ***p < 0.001 when comparing treatment groups with untreated controls. (c) MDA-MB-468 cells were treated with 1 μM AF in the presence or absence of Z-VAD-fmk. Z-VAD-fmk was added to one set of cells 2 hr before adding AF at 0, 2, 4, and 6 hr. The percent caspase activation was determined by a fluorescent spectrophotometer. Data represent the mean of 3 independent experiments performed in duplicate. (d) Western blot analysis was done for cleaved PARP in MDA-MB-468 cells treated with 1 μM AF for the indicated times. GAPDH was used as a loading control. PARP was shown to be cleaved as early as 8 hr. Data shown is a representative study of at least 3 independent experiments with similar results.

Article Snippet: Hoffman modulation phase contrast microscopy Apoptosis was also visualized using an Olympus IX70 inverted microscope equipped with Hoffman modulation phase contrast and images were acquired using a SPOT digital camera system.

Techniques: Microscopy, Staining, Activation Assay, Spectrophotometry, Western Blot

Journal:

Article Title: Aminoflavone induces oxidative DNA damage and reactive oxidative species-mediated apoptosis in breast cancer cells

doi: 10.1002/ijc.23244

Figure Lengend Snippet: Caspases are involved in AF-induced growth inhibition and apoptosis. (a) In the Alamar Blue assay, cells were seeded in 96-well plates and allowed to attach overnight followed by treatment with AF alone or in combination with Z-VAD-fmk for 12 hr. Certain cells were pretreated with 100 μM Z-VAD-fmk for 1 hr prior to 1 μM AF treatment. Growth inhibition was analyzed using a microplate reader. Data are shown as mean of at least 3 independent experiments; bars, SEM where *p < 0.05 when comparing the indicated treatment groups. (b) Hoffman Modulation phase contrast microscopy (×40 magnification) was used to visualize the formation apoptotic bodies. Data for the microscopy studies are representative of at least 3 independent experiments producing similar results. (c) Annexin V-PI staining of cells was used to evaluate apoptosis and determine whether AF-induced apoptosis is caspase dependent. Data is reported as the mean of at least 3 independent experiments; bars, SEM. ***p < 0.001 when comparing the indicated treatment groups.

Article Snippet: Hoffman modulation phase contrast microscopy Apoptosis was also visualized using an Olympus IX70 inverted microscope equipped with Hoffman modulation phase contrast and images were acquired using a SPOT digital camera system.

Techniques: Inhibition, Alamar Blue Assay, Microscopy, Staining

Journal:

Article Title: Aminoflavone induces oxidative DNA damage and reactive oxidative species-mediated apoptosis in breast cancer cells

doi: 10.1002/ijc.23244

Figure Lengend Snippet: Growth inhibition, apoptosis and caspase activation by AF is attenuated by NAC. (a) In an Alamar Blue assay, MDA-MB-468 or T47D cells were seeded in 96-well or 6-cm2 tissue culture plates overnight followed by treatment with AF alone or following 1 hr pretreatment with NAC for 12 hr and 24 hr, respectively. Cells were pretreated with 20 mM NAC for 1 hr prior to AF (1 μM) treatment. Data are represented as mean of at least 3 independent experiments; bars, SEM where *p < 0.05 or **p < 0.01 when comparing the indicated treatment groups, (b) Hoffman Modulation phase contrast microscopy (×40 magnification) was used to determine whether reactive oxygen species might play a role in AF-induced apoptosis. MDA-MB-468 cells received vehicle, AF, NAC or NAC pretreatment followed by AF treatment as described in Materials and Methods. Data shown is representative of at least 3 independent experiments producing similar results. (c) A caspase 3 activity assay was performed to determine whether AF-induced caspase activation leading to apoptosis is dependent on reactive oxygen species formation. Data represent the mean of at least 3 independent experiments; bars, SEM where ***p < 0.001 when comparing the indicated treatment groups. (d) This schematic diagram represents the proposed mechanism by which AF mediates anticancer effects by modulating reactive oxygen species and inducing DNA damage and apoptosis in sensitive cancer cells. AF is converted into a metabolite, likely 3-OH AF which can induce ROS which results in caspase activation and apoptosis. Via a positive feedback loop, caspases can sustain ROS induction leading to DNA damage. Alternatively, ROS can themselves be produced from CYP which can damage DNA and lead to apoptotic responses.

Article Snippet: Hoffman modulation phase contrast microscopy Apoptosis was also visualized using an Olympus IX70 inverted microscope equipped with Hoffman modulation phase contrast and images were acquired using a SPOT digital camera system.

Techniques: Inhibition, Activation Assay, Alamar Blue Assay, Microscopy, Caspase-3 Activity Assay, Produced