PPs protection of human HaCaT keratinocytes from UV-C cytotoxicity
In preliminary experiments, the effect of UV-C radiation at doses of 0.06 J/cm2 on the viability of human keratinocytes was studied at various time intervals after irradiation using the PrestoBlueTM Reagent. As follows from the data in Table 1, UV-C did not have a significant effect on the viability of keratinocytes 4 h after exposure, while after 24 h the number of viable keratinocytes decreases by more than 80 % compared to the non-irradiated control.
Table 1 The effect of UV radiation on the viability of cultured keratinocytes (НаСаT) 4 and 24 h after exposure
Experimental conditions
|
Percentage of viable cells
|
Time after irradiation 4 h
|
Time after irradiation 24 h
|
Sham-irradiated control
|
100.0 ± 14.3
|
100.0 ± 8.3
|
UV-C, 0.06 J/cm2
|
89.7 ± 9.4
|
12.9 ± 3.9***а
|
***а - Р < 0.001 vs sham-irradiated control
In the following experiments, we studied the cytoprotective activity of a number of PPs added to cells at a concentration of 50 µmol/L both before and after UV exposure. In the first line of experiments, cells were preincubated with PPs for 30 min, then the cells were irradiated and incubated in a medium without PPs. In the second case, PPs was added to the medium immediately after irradiation, and irradiated cells were incubated in the presence of PPs.
Table 2 The effect of PPs (50 μmol/L) added to cells before and after UV-C radiation (0.06 J/cm2) on the viability of cultured keratinocytes 24 h after exposure
Experimental conditions
|
Percentage of viable cells
|
PPs added before UV exposure
|
PPs added after UV exposure
|
Sham-irradiated control
|
100.0 ± 13,5
|
100.0 ± 13,5
|
UV-C (negative control)
|
13.1 ± 6.1***а
|
12.8 ± 6.1***а
|
UV-C and acacetin
|
16.4 ± 7.6
|
39.6 ± 7.6***b
|
UV-C and silybin
|
14.7 ± 2.7
|
38.5 ± 7.9***b
|
UV-C and quercetin
|
15.8 ± 6.7
|
28.1 ± 6.7**b
|
***а - Р < 0.0001 vs control; **b - Р < 0,01 и ***b - Р < 0.001 vs UV-C
The data given in Table 2 indicate that PPs added after irradiation significantly increased the number of viable keratinocytes 24 h after exposure compared to UV-C irradiated cells incubated without PPs. The cytoprotective activity of PPs decreased in the following order: acacetin, silybin, resveratrol, quercetin. At the same time, PPs preincubated with cells for 30 min were ineffective.
The cytoprotective effect of acacetin and silybin against UV-C-induced cell death was confirmed by a cell viability test (Live/Dead staining) applied after 18 h. Two fluorescent dyes, AO and EB were used to distinguish between living and dead cells. The fluorescent micrographs shown in Fig. 1 demonstrate that after exposure to UV-C at a dose of 0.06 J/cm2 (Fig. 1b), the total number of cells significantly decreased compared to control samples (Fig. 1a), besides many UV-irradiated cells exhibited intense red fluorescence due to staining with EB, indicating injury of cell membranes. However, incubation of cells in the presence of PPs resulted in an increase in number of living cells (Fig. 1c and 1d).
Effect of acacetin added to cells after irradiation on UV-induced histone H2AX phosphorylation
The protective effect of PPs upon exposure of human keratinocytes to UV-C radiation at a dose of 0.06 J/cm2 may be due to a number of mechanisms, including an increase in the efficiency of DNA repair processes. Therefore, in following experiments, we studied the possible effect of acacetin added to cells after irradiation on UV-induced histone H2AX phosphorylation. This process is one of the earliest events in DNA repair and resulting in the appearance of phosphorylated H2AX within 1 h after UV-C exposure [12]. This time interval was chosen to assay amount of γH2AX by immunofluorescent staining using primary antibodies to γH2AX (pS139). Three separated coverslips for every experimental condition in two different experiments were used. Staining nuclei in a total of nearly 400 cells were visualized and photographed using a fluorescence microscope (Fig. 2).
Median staining intensity and mean area in pixels were measured for every nucleus. Total staining intensity of the nucleus was calculated as the result of multiplying the intensity by the area. The data shown in Table 3 indicate that UV-C irradiation caused activation of H2AX phosphorylation in keratinocytes and increase the amount of γH2AX 1 h after exposure. This results completely coincides with the data obtained earlier by T. Marty et al. [12]. It was also found that in the presence of acacetin, the process of H2AX phosphorylation was significantly accelerated and the amount of γH2AX detected in nuclei of keratinocytes 1 h after UV-C irradiation was almost twice as high as in UV-irradiated cells and four times more than in Sham irradiated control cells.
Table 3 Effect of acacetin (50 µmol/L) added to cells after irradiation on UV-induced histone H2AX phosphorylation measured as described in “Materials and Methods”
Experimental conditions
|
Median staining intensity
|
Median area
|
Median total staining intensity
|
Sham irradiation
UV-C, 0.06 J/cm2
UV-C and acacetin
|
1.0 ± 1.0
2.6 ± 1.6 а
4.2 ± 2.0 b
|
1.0 ± 0.5
0.9 ± 0.4
1.0 ± 0.4
|
1.0 ± 1.4
2.2 ± 1.6 а
3.9 ± 2.9 b
|
*- The data are presented in arbitrary units (AU) normalized to sham irradiation as mean ± SD for the nucleus
а - Р < 0.00000001 vs control; b - Р <0.00000001 vs UV-C
Effect of PPs added to cells after irradiation on UV-induced DNA lesions
It is known that impact of UV-C irradiation on cellular DNA leads to DNA single-strand damage as a result of fast photochemical processes. The cellular response to DNA damage is the activation of repair mechanisms. The data presented in the previous section can be considered as evidence of the ability of PPs and, in particular, acacetin to activate and accelerate reparative DNA synthesis after exposure to UV-C. This phenomenon may result in a decrease in the amount of CPDs and (6-4)-photoproducts in damaged DNA. Consequently, in the following experiments we studied the amount of CPDs and (6-4)-photoproducts in nuclear DNA keratinocytes which were incubated with and without PPs after UV-C irradiation. Among the most common and accuracy methods applied to evaluate the diverse effects of genotoxic agents and to identify the gene-protective effect of potential pharmacological drugs is the comet assay. It was first developed by Ostling and Johanson in 1984 [22]. Alkaline version of the comet assay uses alkaline denaturation surrounding a DNA break to reveal the break (single or double) [23]. This technique we applied to estimate the gene-protective effect of PPs. Irradiated cells were harvested after 2 h cultivation with or without PPs and were treated as described in Materials and methods. Stained slides were observed using a fluorescence microscope and photographed using a digital camera (Fig. 3). Percentage of DNA in the head (undamaged) and the tail (damaged) was scored after pooling the data from three independent experiments (Table 4).
Table 4 Effect of PPs (50 µmol/L) added to cells after irradiation on percentage of DNA in the “comet head” and the “comet tail” 2 h after keratinocytes exposed to UV-С
Experimental conditions
|
Percentage of DNA in head
|
Percentage of DNA in tail
|
Sham irradiation
UV-C, 0.06 J/cm 2
UV-C and quercetin
UV-C and silybin
UV-C and acacetin
|
98.1 ± 0.01
30.0 ± 12.4*** а
44.3 ± 19.7*** б
41.8 ± 16.3*** б
39.7 ± 14.2** б
|
1.9 ± 0.01
70.0 ± 12.4*** а
55.7 ± 19.7*** б
58.2 ± 16.3*** б
60.3 ± 14.2** б
|
***а - Р < 0.0000001 vs Sham irradiation; **б - Р <0.01; ***б - Р <0.001 vs UV-C
According to the data showing in Fig. 3 and Table 4, UV-C irradiation resulted in DNA damage, which was manifested by the increasing the percentage of DNA in the comet tail to 70 % 2 h after exposure. All polyphenols studied were partially able to eliminate DNA lesions as evidenced by the decrease in “comet tails” of damaged cells.
It is common knowledge that the main UV components of terrestrial radiation from sun are UV-A (95 %) and UV-B (5 %) whereas UV-C are completely absorbed by the earth's atmosphere. Therefore, for studying biological effects of solar UV radiation usually used medium and long wavelength UV light (280 to 400 nm). However, cells exposure to UV-C radiation may be useful for studying DNA reparation as a target for testing drug effects. Due to that exposure to UV-C radiation leads to direct photo-damage of DNA, this approach allows excluding the formation of a significant amount of CPDs and 6-4 photoproducts as a result of oxidative damage and therefore, to ignore antioxidant activity of tested compounds. In order to avoid screening effects, cells were irradiated with UV in the absence of PPs.
The cellular response to single-stranded DNA damage is the initiation of cell cycle arrest and DNA repair using an intact complementary strand as a template [1]. However, if complete repair does not occur, intrinsic signals resulting in apoptotic cell death [24]. In our experiments, keratinocytes were irradiated with UV-C at a dose of 0.06 J/cm2 and significant cell death occurring 24 h after. The cells death was interfered by the addition in culture media of PPs after the irradiation. To clarify the possible mechanism underlying the anti-apoptotic effect of PPs we used comet-assay and gamma H2AX staining followed by microscopy. The data received indicate that All PPs were able to decrease the number of radiation-induced photoproducts in the genomic DNA and suggest that this occurs via the acceleration of DNA repair.
In summary, inhibition of cell death without DNA protection may be responsible for the increased incidence of skin tumors after UV irradiation [25, 26]. Oppositely the upregulation of DNA repair pathways resulting in reduced UV-associated mutation. Therefore PPs, which manifested both cell death suppression and upregulation of DNA repair may be suggested as agents for pharmaceutical applications as well as perfect ingredients for solar cosmetic products.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.