Antioxidant and anti-proliferative properties of extracts from heterotrophic cultures of <i>Galdieria sulphuraria</i>

<p>This study explores the possibility to use the extremophilic microalga <i>Galdieria sulphuraria</i> (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. <i>Galdieria</i> was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. <i>Galdieria</i> extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that <i>G. sulphuraria</i> from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.</p

Spheres formation, cytometric analysis and telomerase activity.

<p>(A) Sphere clusters formed by CD34⁺/CD90⁺ cells in semisolid medium after 24 hours (Original Magnification×100); (B) Cytometric analysis on adherent cells for CD90 (80%) and CD34 (80%) antigens and on floating spheres for CD90 (2–3%) and CD34 (95–98%) antigens; (C) Telomerase activity of differentiated endothelial cells (ΔA = 0.160) was significantly reduced (p<0.001) respect to undifferentiated CD34⁺/CD90⁺ cells (ΔA = 0.377)</p

RT-PCR analysis.

<p>(A) Representative figure of RT-PCR showing mRNA transcript expression of CD90, CD34, CD44, CD54, VEGF, Flk-1, on cells in DMEM 10% FBS 7, 15 and 30 days of culture; (B) PPARγ and adiponectin on cells in adipogenic medium at 7 and (C) 30 days of culture.</p

Cell cycle analysis performed using Hoechst 33342 and Ki67 both on CD34 negative and positive cells.

<p>(A) Hoechst 33342 analysis performed CD34⁻ cells: G₀G₁ phase (98%), S phase (0,65%) and G₂M phase (0,50%); (B) Ki67 analysis performed CD34- cells: Ki67 (10%); (C) Hoechst 33342 analysis performed CD34⁺ cells: G₀G₁ phase (84%), S phase (5%) and G₂M phase (10%); (D) Ki67 analysis performed CD34+ cells: Ki67 (85%).</p

Adipogenic differentiation.

<p>(A) ASCs in DMEM 10% FBS exhibit a fibroblast-like morphology (Original magnification 100×); (B) ASCs in adipogenic medium exhibit an adipocyte morphology (Original magnification 100×); (C) CD34⁺/CD90⁺ cells in DMEM 10% FBS showing negativity for adiponectin by immunohistochemistry (Original magnification 400×); (D) CD34⁺/CD90⁺ cells in adipogenic medium showing positivity for adiponectin by immunohistochemistry (Original magnification 100×); (E) CD34⁻/CD90⁻ cells in DMEM 10% FBS showing negativity for adiponectin by immunohistochemistry (Original magnification 100×); (F) CD34⁻/CD90⁻ cells in adipogenic medium showing negativity for adiponectin by immunohistochemistry (Original magnification 400×).</p

Representative flow cytometry analysis performed on CD34⁺/CD90⁺ cells at day 30 from sorting.

<p>A significant number of ASCs are clearly positive for endothelial markers, including CD90 (97%), CD44 (90%), CD54 (90%), VEGF (70%), CD133 (18%), and Flk-1 (60%). Control PE-conjugated and FITC-conjugated isotypes were negative.</p

Colony-forming efficiency of CD133⁺ cells versus CD133− population^a in SAOS2, MG63 and U2OS cell lines.

a<p>Results are the mean±standard devition of three experiments from different cases.</p>b<p>Represents the number of colonies with respect to the number of wells plated in experiments.</p>c<p>Ratio between the percentage of colonies formed by CD133+ versus CD133− cells.</p

Side population analysis.

<p>(A) Cytometric analyses of the side population. The CD133⁺ fraction includes a small subset (0.97%), expressing the characteristic profile of a side population at FACS. (B) ABCG2 expression in SAOS2 cell line, showing an evident positivity; the grey line indicates the isotype control.</p

CD133 expression in adherent cells and floating spheres.

<p>(A) Immunohistochemical analyses on adherent cells and (B) floating spheres showing the presence of CD133 antigen (<u>arrows</u>). (Original Magnification. ×100); (C) Immunofluorescence analysis on SAOS-2 for CD133 PE, cytoskeleton is stained with phalloidin-FITC, nucleus with DAPI (Original Magnification. ×400); (D) Immunoflurescence analysis on SAOS-2 spheres for CD133 PE after 24 hours in adhesion. (Original Magnification ×200); (E) Confocal analyses on adherent cells and (F) floating spheres confirming the presence of the CD133 antigen. (Original Magnification. ×400).</p

Cytometric analyses for CD133 and OCT3/4 on sarcospheres in SAOS2, MG63 and U2OS.

<p>The blue line indicates isotype controls, red and green lines indicate the expression of CD133 at the 4^th and 6^th cell passage, respectively. In the histograms, OCT3/4 expression is analyzed at the 6^th cell passage; the green line indicates isotype controls. Sarcospheres both in CD133 and OCT3/4 are strongly positive.</p