Evaluation of the anti-cancer potential of <i>Mahonia aquifolium</i> extracts via apoptosis and anti-angiogenesis

Abstract

&lt;p class="Abstract"&gt;The cytotoxicity of &lt;em&gt;Mahonia aquifolium&lt;/em&gt; ethanol and water extracts was examined using MTT test. The morphological changes were analyzed by fluorescence microscopy. Cell cycle distribution and possible activation of caspase-dependent pathway of cell death were assessed by flow cytometry. The effects of ethanol and water extracts on migration of endothelial EA.hy926 cells were analyzed by&lt;em&gt; in vitro&lt;/em&gt; scratch assay and inhibition of angiogenesis was detected using tube formation assay. Both extracts demonstrated cytotoxic effects on cancer cell lines with very high selectivity. Morphological evaluation indicated apoptosis. These results were confirmed with cell cycle analysis, where there was accumulation of cancer cells in the subG1 phase. Ethanol and water extracts induced a caspase-dependent apoptosis in HeLa cells through activation of caspase-3 and caspase-8. Both extracts showed the ability to inbibit the migration of EA.hy926 cells and initial steps of angiogenesis. In addition, ethanol extract exerted significant anti-angiogenic effect.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Video Clip:&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;a href="https://www.youtube.com/v/_b1LobagxyA"&gt;Cell seeding&lt;/a&gt;: 2 min 26 sec&lt;/p&gt;&lt;p&gt;&lt;a href="https://www.youtube.com/v/LZrwdyvFAyI"&gt;4 hours after treatment of MTT-SDS applied&lt;/a&gt;: 54 sec&lt;/p&gt;&lt;p&gt;&lt;a href="https://www.youtube.com/v/a6Dshdsg8TY"&gt;72 hours after treatment MTT applied&lt;/a&gt;: 1 min 8 sec&lt;/p&gt;&lt;p&gt;&lt;a href="https://www.youtube.com/v/RE_SH-CauRU"&gt;Day 2 treatment&lt;/a&gt;: 1 min 32 sec&lt;/p&gt;&lt;p&gt;&lt;a href="https://www.youtube.com/v/l4E1y0lgrAE"&gt;Measuring absorbance&lt;/a&gt;: 9 sec &lt;/p&gt;

Seasonal variation in biopharmaceutical activity and fatty acid content of endemic Fucus virsoides algae from Adriatic Sea

“…Seeding densi-ties for each cell line were: 2000, 7000, 5000, 5000 and 5000 cells per well, respectively. Cells were allowed to adhere overnight and treated with serial dilutions of extracts and fractions ranging from 12.5 to 200 µg/mL, according to the protocol described previously (16). The final concentrations of DMSO to which the cells were exposed were non-toxic, lower than 0.5%.…”

“…The effects of the extracts and fractions on cell survival after 72 h of treatment (for HeLa, LS-174T, A549, and MRC-5), and 24 h treatment (for EA.hy 926 cells), were determined by MTT cell survival assay, described in detail previously (16). IC 50 was defined as the concentration of an agent that inhibited cell survival by 50% compared to control.…”

“…HeLa cells were seeded into 6-well plates (200000 cells per well) and after 24 h they were treated with IC 50 and 2 × IC 50 concentrations of investigated extracts and fractions for 24 h. After incubation, the cells were collected by trypsinization, fixed in 70% ethanol on ice and stored at -20 O C for one week (16). The cells were washed and incubated with RNaseA (100 µg/mL) at 37 O C for 30 min.…”

“…After 24 h of treatment, the cells were stained with a mixture of acridine orange and ethidium bromide dyes (3 µg/mL acridine orange and 10 µg/mL ethidium bromide in phosphate-buffered saline (PBS). Photo-micrographs were taken under a fluorescence microscope ñ Carl Zeiss PALM MicroBeam with AxioObserver.Z1 using AxioCamMRm (filters Alexa 488 and 568), as previously described (16).…”

“…EA.hy926 cells were seeded into 24-well plate. Confluent cell monolayers were formed after 24 h and scarped with a p200 pipette tip to create a straight central scratch line, as described earlier (16). After washing with nutrient medium cells were treated with subtoxic concentrations (IC 20 ) of extracts and fractions for 24 h. IC 20 concentration is the concentration that inhibits 20% of cell survival, and values for fall samples were 88.0 µg/mL, 94.3 µg/mL, 72.9 µg/mL, 122.1 µg/mL and for summer 147.2 µg/mL, 156.6 µg/mL, 160.9 µg/mL, 161.8. µg/mL for dichloromethane-methanol, petroleumether, ethyl-acetate, and n-butanol extracts and fractions respectively.…”