174 JOURNAL OF COSMETIC SCIENCE
increasing the risk of genetic alterations. Hence, skin protection against UV light can be
considered preventive medicine, as there becomes a need toward preventing short-term
(erythema, inflammation) and long-term (cancer, aging) effects of exposure to this harmful
radiation. Epidermal keratinocytes are a major target of radiation-induced damage as they
make up most epidermal cells.
In the last decades, the public has become widely aware of the risks of sustained exposure
to sunlight, and the use of sun-protection products has significantly increased. Diverse
natural products have been used to counteract UV radiation–induced skin damage
through different approaches. One is the use of sunscreens, which prevent high-energy
photons from reaching sensitive biological targets, e.g., nuclear DNA these substances
possess the ability to absorb photonic energy, thereby preventing biological substrates
from doing it. Another possibility is to use substances that do not act as a sunscreen
but minimize injury though alternative mechanisms, e.g., attenuating oxidative or DNA
damage. These substances do not necessarily need to be applied topically, but they can
also be administered orally.5
Our group has focused on the beneficial effects of a hydrophilic extract from the leaves
of the fern Polypodium leucotomos (PLE, Fernblock®). PLE has a remarkable safety profile
(Nestor et al., 2015), has a function as a barrier (it absorbs UV photons), and acts as
a biological modifier.6,7 However, its main beneficial effect is due to its extraordinary
antioxidant ability and protective cellular effects. These include improved myeloid
(Langerhans) cell survival upon UV irradiation, decreased trans–urocanic acid
isomerization and increased DNA repair enzyme activity.8–11 PLE is currently used in
topical and oral sunscreen formulations.12 It also counteracts other deleterious effects of
UV irradiation, e.g., photoaging and photoimmunosuppression.13,14 Its mechanism of
action seems multipronged, but its antioxidant capability plays a fundamental role in its
beneficial effects.9
Although the above beneficial effects are well documented, the genetic protective
mechanisms of PLE in the keratinocyte biology have not been addressed yet. Unrepaired
DNA damage blocks proliferation and induces differentiation of keratinocytes of the diverse
stratified epithelia of the skin or head and neck.15–17 This response is controlled by mitotic
checkpoints.16,18–20 The differentiation response protects keratinocytes from apoptosis even
upon moderate UV irradiation.21 The G2 phase of the cell cycle, prior to mitosis, is a major
phase for DNA repair. Upon DNA damage, G2 checkpoints halt the cycle to ensure that
DNA is repaired before cell division.22 Multipotent stem cells within the basal layer of the
epidermis maintain the tissue and are targets of genetic insult and aging.23,24 These cells
are not totipotent but are capable of replenishing the tissue and giving rise to different
progenitors.
We have investigated the effects of treating primary human keratinocytes with PLE on
DNA damage and the cell cycle before or after UV irradiation. Interestingly, epidermal
keratinocytes treated with PLE were retarded in growth but conserved a greater capacity to
amplify after treatment. We have investigated the molecular mechanisms underlying this
protective effect. A “soft arrest” in G2/M upon PLE treatment might allow cells to better
repair their DNA before undergoing mitosis. PLE appears to achieve this by enhancing
cellular DNA repair signals. This mechanism might contribute to preserve genome
integrity and the proprieties of epidermal stem cells face to DNA replication stress or UV
irradiation. We discuss the implications for skin cancer and aging.

175 POLYPODIUM LEUCOTOMOS EXTRACT
RESULTS
PLE SLOWS DOWN GROWTH AND PROTECTS THE AMPLIFYING CAPACITY OF HUMAN
EPIDERMAL KERATINOCYTES
We cultured freshly isolated keratinocytes from human epidermis in Rheinwald
conditions.25 In these conditions, cells proliferate, stratify, and differentiate. To investigate
the effect of PLE on the clonal growth capacity of epidermal stem cells, we performed
clonogenicity assays with human isolated primary keratinocytes of the skin. These assays
at low density allow monitoring the growth of epidermal stem cells since they are the
only cells capable to give rise to large proliferative colonies.26Proliferative progenitors
are committed to differentiate after a small number of divisions and give rise to small
differentiated colonies. Primary keratinocytes cultured in the presence of PLE for 8 days
(0.8 or 1.6 mg/ml) displayed a significantly greater reduction in the size of the colonies
than controls (Figure 1A). When we examined the cultures after incubation with PLE for
8 days, we observed that cells were slightly larger and less densely packed than controls
(Figure 1B). The smaller size of the colonies upon PLE might be caused by a toxic effect or
by retarded growth. To answer this question, we plated cells for clonogenicity assays after
treatment, in the absence of PLE, to test for the later effects on the capacity of stem cells.
Interestingly, PLE-treated keratinocytes displayed a significant increase in the number of
colonies (Figure 1C).
PLE DELAYS PROGRESSION OF KERATINOCYTES THROUGH THE G2 PHASE OF THE CELL CYCLE
AND INDUCES DNA REPAIR SIGNALS ΓH2AX AND P53 INDEPENDENTLY OF DNA DAMAGE
The growth inhibitory effect of PLE suggested that it was acting on the keratinocyte
cycle. Therefore, we examined the effect on the cell cycle. As before, we treated human
keratinocytes with PLE (0.8 mg/ml) for 8 days and determined their DNA content by
flow cytometry. We observed a striking change in the cell cycle upon PLE. G1 was
significantly diminished and G2/M was augmented (Figure 2A). We found a similar
accumulation of cells in G2/M after various periods of treatment with PLE, from 5 to 9
days (not shown). Considering that cells proliferated more slowly, this would not indicate
a cell cycle activation but rather, a delay of progression through the G2/M transition.
We observed no signs of apoptotic sub-G1 cells in the DNA content analyses, further
indicating that the growth inhibitory effect of PLE was due to a slower cell cycle. An
increase in forward scatter and side scatter was also observed within the proliferative
region, consistent with the morphology displayed in Figure 1 and with the typical cell
size increase in G2/M due to the cellular growth that takes place prior to cell division
(Figure 2B).20,26
Since keratinocytes were more clonogenic after the PLE treatment, we investigated the
expression of γH2AX by flow cytometry, a central signal in the induction of the DNA repair
cascade.27 Interestingly, upon a similar PLE treatment as before, we observed a significant
increase in the phosphorylation of H2AX (γH2AX Figure 3A and B). Since γH2AX
induces the G2/M checkpoint to allow DNA repair, in part via the p53, we analyzed the
expression of this transcription factor by Western blotting and immunofluorescence (Figure
3C and D). PLE treatment significantly induced the expression of p53 to levels comparable
to those induced by treatment with the genotoxic drug Doxorubicin (Figure 3C).