*3.4. Treatment with Sea-Buckthorn Oil Following UVA Irradiation Does Not Mitigate the Deleterious Effect on Dysplastic Cells*

As sea-buckthorn oil is reported to have protective effects against irradiation, we tested the effect of UVA irradiation on normal and dysplastic keratinocytes adherence using a real-time impedance reading system. This investigation allowed for the quantification of cell adherence (e.g., required for re-adhesion of daughter-cells after cell division) and of cell proliferation by calculation of doubling times using the xCELLigence platform (Figure 4). Consistent with videomicroscopy observations, oil treatment impairs cell adhesion and fails to mitigate UVA irradiation effects. Doubling times analysis confirmed MTS data regarding stimulation of cell viability with the selected dilution of sea-buckthorn seed oil. Notably, oil treatment on irradiated cells significantly decreased doubling times, which translates into increased proliferation rate of cells.

**Figure 4.** Cell adhesion and proliferation of normal and dysplastic keratinocytes, treated with sea-buckthorn seed oil, in the presence or absence of UVA irradiation. Each point on graphs represents the average cell index of triplicates, recorded every 15 min for 24 h. Bars represent average of triplicates with SD. One-Way Anova was used to assess statistical significance (\*\* *p* < 0.01, \*\*\*\* *p* < 0.0001).

#### *3.5. UVA Irradiation Does Not Affect Uptake of Lipids into Normal and Dysplastic Keratinocytes*

Next, we tested whether irradiation affects oil uptake into cells. Keratinocytes use lipids to seal the epidermis from the outer environment by deposition of sphingolipids in the extracellular space [18]; therefore, a normal degree of lipid inclusion is to be expected in non-treated cells. Both normal and dysplastic cells uptake presented increased lipid load following oil treatment. However, unlike normal cells, where only some cells are loaded with lipid inclusions, all dysplastic cells have a degree of lipid load. Irradiation did not significantly affect the upload of lipids in both normal and dysplastic cells, but dysplastic cells showed a more uniform lipid uptake (Figure 5).

**Figure 5.** Oil uptake in irradiated and non-irradiated cells. Irradiated and non-irradiated cells were treated with seabuckthorn seed oil (dilution 1/8000) for the indicated periods of time. Non-treated, non-irradiated cells were used as controls. Lipid inclusions were stained with Oil Red O.

#### *3.6. The Fatty Acid Translocator CD36 Is Expressed in Dysplastic, but Not Normal Keratinocytes*

Long-chain FA used by cells to synthesize phospholipids and sphingolipids do not easily diffuse through cell membranes and are more efficiently up-taken with the help of fatty acid translocators, such as CD36/SR-B2. Furthermore, CD36/SR-B2 was shown to be involved in the tumorigenesis of aggressive, metastatic cancers (reviewed in [19]). Using two different methods, we investigated the expression of CD36 in normal and dysplastic keratinocytes following UVA irradiation. The expression of CD36 in normal keratinocytes is undetectable in both IF (data not shown) and WB, and it appears to be slightly induced by irradiation. In contrast, dysplastic keratinocytes express a stronger signal for CD36, in both control and irradiated cells (Figure 6).

**Figure 6.** CD36 expression in normal (HEKa) and dysplastic (DOK) keratinocytes after UVA irradiation. Expression of CD36 in both HEKa and DOK cells was assessed by confocal immunofluorescence and confirmed by Western blot. In basal conditions, CD36 expression in normal keratinocytes is undetectable with the selected methods. Its expression increases in dysplastic cells, as well as following UVA irradiation.

#### **4. Discussion**

Our manuscript compares the effect of sea-buckthorn seed oil on normal and dysplastic human keratinocytes, starting from the premise that normal skin contains a mixture of both cells, whose proportion changes with age. It was demonstrated that sea-buckthorn fatty acids have the ability to improve the post-inflammatory response resulting from deleterious UV exposure. Moreover, they could alleviate the effects of sun burns, support regenerative processes of the skin and appease irritation [5]. UVA exposure was reported to affect these cell populations differently in terms of migration and proliferation. It was previously demonstrated that dysplastic cells are less able to spread across a denuded area than normal keratinocytes, and were more affected than the latter by UVA treatment [17]. In terms of cell proliferation, UVA exposure induced cell-cycle arrest for normal keratinocytes [20,21]; this outcome, reviewed in [22], may lead to an increased doubling time of a cell population, a finding which also was observed in our real-time, impedance-reading experiments. Dysplastic cells do not exhibit this protective effect; they are more prone to proliferate under UV irradiation, and the seed-oil treatment further aggravates this noxious behavior.

One putative mechanism for increased dysplastic cell proliferation following seed -il treatment could be CD36-mediated lipid uptake. Fatty acid transporters, including CD36, are overexpressed in tissues with an increased fatty acid metabolism [23]. FA are able to activate peroxisome proliferator-activated receptors (PPARs) [24], transcription factors which further activate gene transcription of CD36 [25]. It was previously shown that tumor cells modify their lipid metabolism [15,16] to favor cell survival and proliferation. A study by Pascual et al. revealed that CD36+ cells react to dietary lipids and rely on lipid metabolism for metastatic potential. In their study, CD44 bright cells isolated from human oral carcinomas exerted a distinctive ability to overexpress both the fatty acid receptor CD36 and lipid metabolism genes, thus accelerating the initiation of metastasis [26].

Various studies in the literature shed light on the potential relationship between an impaired lipid metabolism and different skin conditions, but the extent to which fatty acid transporters are involved in directing fatty acids to keratinocytes, altering the fate of lipid metabolism, remains an open question [23]. As far as the current literature goes, normal keratinocytes do not require CD36 for their metabolism; it is only transiently expressed in pathological circumstances such as wounds [27], infectious [28] and autoimmune [29,30] cutaneous diseases and tumor-related pathologies [31]. A stable CD36 expression can be a driver for increased lipid metabolism, which can fuel a possible malignant transformation.
