Treatment with Modified Extracts of the Microalga Planktochlorella nurekis Attenuates the Development of Stress-Induced Senescence in Human Skin Cells.

More recently, we have proposed a safe non-vector approach to modifying the biochemical profiles of the microalga Planktochlorella nurekis and obtained twelve clones with improved content of lipids and selected pigments and B vitamins and antioxidant activity compared to unaffected cells. In the present study, the biological activity of water and ethanolic extracts of modified clones is investigated in the context of their applications in the cosmetic industry and regenerative medicine. Extract-mediated effects on cell cycle progression, proliferation, migration, mitogenic response, apoptosis induction, and oxidative and nitrosative stress promotion were analyzed in normal human fibroblasts and keratinocytes in vitro. Microalgal extracts did not promote cell proliferation and were relatively non-cytotoxic when short-term treatment was considered. Long-term stimulation with selected microalgal extracts attenuated the development of oxidative stress-induced senescence in skin cells that, at least in part, was correlated with nitric oxide signaling and increased niacin and biotin levels compared to an unmodified microalgal clone. We postulate that selected microalgal extracts of Planktochlorella nurekis can be considered to be used in skin anti-aging therapy.

WE1 to WE12) are shown. Control clone water extract is denoted as CWE. ERK1/2 activity was measured using Muse ® Cell Analyzer and Muse ® MAPK Activation Dual Detection Kit.

Supplementary Figure 2.
Extract-mediated effects on cell migration. Scratch wound healing assay. BJ cells (a) and HEK cells (b) were treated with 100 µg/ml water extracts for up to 72 h after wounding. Cell migration was evaluated under an inverted microscope. Representative microphotographs are shown. Scale bars 500 μm, objective 4x.

Supplementary Figure 3.
Pro-senescence activity of microalgal extracts in BJ cells (a, 100 µg/ml water extracts and 100 µg/ml ethanolic extracts) and HEK cells (b, 100 µg/ml water extracts and 1 µg/ml ethanolic extracts). The effects of water extracts (WE, twelve modified clones from WE1 to WE12) and ethanolic extracts (EE, twelve modified clones from EE1 to EE12) are shown.
Control clone water extract is denoted as CWE and control clone ethanolic extract is denoted as CEE. Senescence-associated β-galactosidase activity. Representative microphotographs are shown. Scale bars 100 μm, objective 20x. To emphasize extract action, a red horizontal line is added. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the control (ANOVA and Dunnett's a posteriori test).

Supplementary Figure 4.
Extract-mediated changes in BJ (a) and HEK cell number (b) after 2 h stimulation with hydrogen peroxide and subsequent cell culture for 7 days in the presence of water (left) and ethanolic extracts (right), and the effect of 24 h treatment with water (left) and ethanolic (right) extracts on BJ (c) and HEK cell number (d) after subsequent cell culture for 7 days without microalgal extracts. Cell number was analyzed using TC10 ™ automated cell counter.
To emphasize extract action, a red horizontal line is added. The effects of water extracts (WE, twelve modified clones from WE1 to WE12, left) and ethanolic extracts (EE, twelve modified clones from EE1 to EE12, right) are shown. Control clone water extract is denoted as CWE and control clone ethanolic extract is denoted as CEE. Bars indicate SD, n = 3. (a, b) Cell number after 2 h stimulation with hydrogen peroxide is considered as 100%. *** p < 0.001, * p < 0.05 compared to hydrogen peroxide treatment (ANOVA and Dunnett's a posteriori test).

Supplementary Figure 5.
Preliminary analysis of anticancer activity of water (a, 100 µg/ml) and ethanolic (b, 100 µg/ml) extracts against MDA-MB-231 breast cancer, U-2 OS osteosarcoma and U-251 MG glioblastoma cells. BJ fibroblasts were used as control normal human cells. The effects of water extracts (WE, twelve modified clones from WE1 to WE12) and ethanolic extracts (EE, twelve modified clones from EE1 to EE12) are shown. Control clone water extract is denoted as CWE and control clone ethanolic extract is denoted as CEE. To emphasize extract action, a red horizontal line is added. Extract-mediated changes in metabolic activity (MTT assay) of cancer and normal cells were investigated. Metabolic activity at standard growth conditions