Proanthocyanidins Inhibit Photocarcinogenesis through Enhancement of DNA Repair and Xeroderma Pigmentosum Group A–Dependent Mechanism

Grapes (Vitis vinifera) are one of the most widely consumed fruits in the world. Grapes are rich in polyphenols and approximately 60% to 70% of grape polyphenols exist in the grape seeds. The grape seeds are available as byproducts of the industrial production of grape juice and wine. The seeds contain a larger fraction of proanthocyanidins, which are composed of dimers, trimers, tetramers, and oligomers of monomeric catechins or epicatechins (1–3). These grape seed proanthocyanidins (GSP) have been shown to have antioxidant (4, 5), antimutagenic, antiinflammatory, and anticarcinogenic (6, 7) activities. GSPs are cytotoxic to various cancer cell lines of different organs (8–10) with no apparent toxic effects on normal cells including human keratinocytes (4, 11). We have shown previously that supplementation of the diet with GSPs inhibits the growth and development of ultraviolet (UV) radiation-induced skin tumors in SKH-1 hairless mice (7). However, the molecular mechanism underlying this inhibitory effect of dietary GSPs is not clearly established.

Cutaneous malignancies, including melanoma and nonmelanoma skin cancers, are a major burden on the health care system, and the incidence of cutaneous malignancy is equivalent to the incidence of malignancies in all other organs combined (12). Exposure of the skin to solar UV radiation is one of the major environmental hazards that induces oxidative stress, inflammation, suppression of immune system, DNA damage, and gene mutation. Collectively, all these events lead to the development of various skin diseases including the risk of cutaneous malignancies (13, 14).

UV irradiation induces DNA damage predominantly the formation of cyclobutane pyrimidine dimers (CPD). UV-induced CPDs have been recognized as a molecular trigger for the initiation of photocarcinogenesis (15–17). Reduction or repair of CPDs through application of DNA repair enzymes considerably reduces the risk of photocarcinogenesis (17, 18). Among the various UV-induced DNA repair mechanisms, nucleotide excision repair (NER) represents the most versatile DNA repair pathway in cells (19). NER recognizes bulky, helix-distorting changes, such as CPD. Xeroderma pigmentosum complementation group A (XPA) gene plays an indispensable role in the NER pathway (20). Patients with XPA-deficiency have a high incidence of skin cancer at an early age on sun-exposed skin caused by loss of the ability to repair UV-induced DNA damage (21).

Here we conducted the studies to determine whether dietary GSPs could prevent ultraviolet B (UVB)-induced skin tumor development through rapid repair of UVB-induced DNA damage and to assess potential mechanisms of this effect. We hypothesized that the rapid repair of UVB-induced DNA damage by dietary GSPs is mediated through the enhanced levels of NER genes, postulating further that DNA repair mechanism by GSPs is mediated through the enhanced levels of interleukin (IL)-12, which has been shown to have DNA repair ability and antitumor activity. Furthermore, if this is the case, dietary GSPs would be unable to repair UVB-induced DNA damage and photocarcinogenesis in IL-12 knockout (IL-12 KO) mice.

Female C3H/HeN mice (6–7 weeks old) were purchased from Charles River Laboratory). The IL-12p40 KO mice on C3H/HeN background were generated and bred in our Animal Resource Facility, as described previously (22). All mice were maintained under standard conditions of a 12-hour dark/12-hour light cycle, a temperature of 24°C ± 2°C and relative humidity of 50% ± 10%. The mice were given control AIN76A diet with or without supplementation with GSPs and drinking water ad libitum throughout the experiment. The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.

The antibodies specific for CPDs were purchased from Kamiya Biomedical Co. The manufacturer-supplied standardized real-time PCR primers for NER genes (XPA, XPC, RPA1, and DDB2), and β-actin were obtained from the SuperArray BioScience Corp.

The GSPs were obtained from the Kikkoman Corporation, and the chemical composition has been described earlier (7). Experimental diets containing GSPs (0.5%, w/w) were commercially prepared in a pellet form in the AIN76A powdered control diet by TestDiet (Richmond) using the GSPs that we provided for this purpose. Dietary administration of GSPs was started at least 2-week before UVB-irradiation of mice and continued until the termination of the experiment.

Mice were UVB-irradiated as described previously (5, 7). Briefly, the clipper-shaved dorsal skin was exposed to UV radiation from a band of four FS20 UVB lamps (Daavlin, UVA/UVB Research Irradiation Unit) equipped with an electronic controller to regulate UV dosage. The UV lamps emit UVB (280–320 nm; ≈80% of total energy) and ultraviolet A (UVA) (320–375 nm; ≈20% of total energy), with ultraviolet C (UVC) emission being significantly less (<1%). The majority of the resulting wavelengths of UV radiation are in the UVB (290–320 nm) range with peak emission at 314 nm.

The IL-12 KO mice on C3H/HeN background and their wild-type (WT) counterparts (C3H/HeN) were divided into three treatment groups with 20 mice in each group. These groups of mice included a control group (non-UVB and non-GSPs), a group that was exposed to UVB in the absence of treatment with GSPs, and a group treated with dietary GSPs prior to UVB irradiation and continued till the termination of the experiment. The photocarcinogenesis protocol employed has been described previously (7). Briefly, the shaved backs of the mice were irradiated with UVB (180 mJ/cm²) three times per week for a total period of 38 weeks. The backs of the mice were shaved using clippers if hairs grown on the skin during the photocarcinogenesis experiment.

UVB-irradiated back of the mice was examined on a weekly basis to check for the growth of papillomas or tumors. At the termination of the experiment, the dimensions of all the tumors on each mouse were recorded. Tumor volumes were calculated using the hemiellipsoid model formula: tumor volume = 1/2 (4π/3) (l/2) (w/2) h, where l = length, w = width, and h = height.

Immunohistochemical detection of CPD⁺ cells in the skin samples was performed using a procedure described previously (23, 25). Briefly, frozen skin sections (5-μm thick) were thawed, and nuclear DNA was denatured, followed by neutralization in 100 mmol/L Tris–HCl (pH 7.5) in 70% ethanol. After blocking nonspecific binding, sections were incubated with antibody specific for CPDs. Bound anti-CPD antibody was detected by incubation with biotinylated goat-anti-mouse IgG1 followed by peroxidase-labeled streptavidin. After washing, sections were incubated with diaminobenzidine and counterstained with either hematoxylin and eosin or methyl green. CPD⁺ cells were detected and photographed under an Olympus BX41 microscope fitted with a Q-Color-5 digital camera.

Epidermal genomic DNA was isolated following the standard procedures, and dot-blot analysis was performed as detailed previously (23, 24). Briefly, genomic DNA (500 ng) was transferred to a positively charged nitrocellulose membrane by vacuum dot-blotting (Bio-Dot apparatus, Bio-Rad) and fixed by baking the membrane for 30 minutes at 80°C. After blocking the nonspecific binding sites, the membrane was incubated with the antibodies specific to CPDs for 1 hour at room temperature. After washing, the membrane was incubated with HRP-conjugated secondary antibody. The circular bands of CPDs were detected using an enhanced chemiluminescence detection system. The genomic DNA was tested at least from five mice/group independently.

XPA-deficient and XPA-proficient human fibroblasts were obtained from the Coriell Institute for Medical Research. The XPA-deficient fibroblasts were obtained from patients suffering from xeroderma pigmentosum group A disease. These fibroblasts were authenticated by the Coriell Institute for Medical Research and supplied for only research purpose. The fibroblasts were cultured in a modified Eagle medium with Earle's salts supplemented with 2 mmol/L l-glutamine, 10% fetal bovine serum, and maintained in an incubator at 37°C in a humidified atmosphere of 5% CO₂. The cells were exposed to the UVB radiation (150 J/m²) through PBS with or without pretreatment with GSPs (20 μg/mL) for 3 hours. The UVB-induced CPD⁺ cells were detected using a protocol described previously (25). Briefly, after UVB irradiation, cells were harvested and cytospun (≈1 × 10⁵ cells/slide). Remaining procedure is similar to tissue staining of CPDs. CPD⁺ cells were counted under the Olympus BX41 microscope in 5 to 6 different fields, and the data are presented as the mean of the percentage of CPD⁺ cells ± SD from two separate experiments.

The epidermal RNA was extracted from the mouse skin samples using TRIzol reagent (Invitrogen), as described (24). The mRNA expression of NER genes, such as XPA, XPC, RPA1, and DDB2, in skin samples was determined using real-time polymerase chain reactions (PCR). For the mRNA quantification, complementary DNA (cDNA) was synthesized using 3 μg RNA through a reverse transcription reaction (iScript cDNA synthesis kit, BIO-RAD). Using SYBR Green/Fluorescein PCR master mix (SuperArray Bioscience Corporation), cDNA was amplified using real-time PCR with a BioRad MyiQ thermocycler and SYBR green detection system (BioRad). Samples were run in triplicate to ensure amplification integrity. Manufacturer-supplied (SuperArray, Bioscience Corporation) primer pairs were used to measure the expression levels of NER genes The standard PCR conditions were 95°C for 15 min, then 40 cycles at 95°C, 30 seconds; 55°C, 30 seconds; and 72°C, 30 seconds, as recommended by the manufacturer. The expression levels of genes were normalized to the expression level of the β-actin mRNA in each sample. The threshold for positivity of real-time PCR was determined based on negative controls. For mRNA analysis, the calculations for determining the relative level of gene expression were made using the cycle threshold method.

Tumor incidence in the UVB and GSPs and UVB treated groups was compared using the χ² test. Tumor multiplicity data were analyzed using the Wilcoxon rank sum test, and tumor volume was statistically analyzed using ANOVA followed by Tukey's post hoc test. A simple ANOVA followed by an appropriate post hoc test was used to calculate statistical significance of the data obtained from tumor volume and the number of CPD⁺ cells in different treatment groups. A P value < 0.05 was considered significant.

In earlier studies, dietary supplementation of GSPs at the level of 0.5% (w/w) significantly inhibited UV-induced skin tumor development (7); therefore, we used 0.5% GSPs in diet for all mice experiments for mechanistic studies. The shaved backs of C3H/HeN mice were exposed to UVB (60 mJ/cm²) with and without the treatment of mice with GSPs. Mice were sacrificed either immediately (≈30 min) or 48 hours later; skin samples were collected and subjected to immunohistochemical detection of CPD⁺ cells. In skin samples obtained immediately after UV exposure, no differences in the staining pattern of CPDs were observed whether or not the mice were treated with GSPs (Fig. 1A). This observation eliminated the speculation that dietary GSPs might have a filtering effect on UV radiation. However, in skin samples collected at 48 hours after UVB exposure, the number of CPD⁺ cells was significantly lower (P < 0.001) in the GSP-treated mice than the mice that have not received GSPs in diet but were exposed to UVB. A summary of CPD⁺ cells in different treatment groups is shown in Fig. 1B. The CPD⁺ cells were not detectable in non-UVB-exposed skin, whether or not they were treated with GSPs (data not shown).

The effect of GSPs on UVB-induced DNA damage was further verified using dot-blot analysis of epidermal genomic DNA isolated from the skin samples from UVB-exposed skin with and without treatment of GSPs. As shown in Fig. 1C, there was no significant difference in the dot-blot pattern of CPDs between skin samples obtained immediately after UVB exposure from UVB-exposed mice whether or not they were treated with GSPs. In samples obtained 48 h after UVB exposure, the intensity of the dot-blot was markedly lower in the GSP-treated mice than the non–GSP-treated control mice. The genomic DNA samples obtained from the groups of mice that were not exposed to UV were negative in the dot-blot assay (data not shown).

As IL-12 has the ability to enhance repair of UVB-induced DNA damage in mouse skin, and we have found that GSPs enhance the levels of IL-12 in UVB-irradiated mice (26), we evaluated whether IL-12 is required for DNA repair by GSPs in UVB-exposed skin. For this purpose, the shaved backs of IL-12 KO and WT mice were exposed to UVB (60 mJ/cm²) with and without pretreatment of mice with diet supplemented with GSPs, and mice were sacrificed 48 h after UVB exposure; skin samples were collected and subjected to immunohistochemical analysis of CPD⁺ cells. In skin samples obtained at 48 hours after UVB exposure, the numbers of CPD⁺ cells were significantly less in the GSP-treated WT mice than the control group that were not treated with dietary GSPs but were exposed to UVB (P < 0.001). In contrast, the numbers of UVB-induced CPD⁺ cells in the skin of IL-12 KO mice that had been treated with GSPs did not significantly reduced from that in the IL-12 KO mice that had not been treated with GSPs (Fig. 2A and 2B).

To further verify the role of IL-12 in the GSP-mediated repair of UVB-induced DNA damage, the shaved backs of GSP-treated IL-12 KO mice were exposed to UVB (60 mJ/cm²) with one group of mice receiving endotoxin-free rIL-12 (1000 ng/100 μL PBS) subcutaneously 3 hours before UVB irradiation. Mice were sacrificed 48 hours later, skin samples collected and subjected to the immunohistochemical analysis of CPD⁺ cells. Summary of immunohistochemical analysis revealed that the number of CPD⁺ cells in the GSP-treated group (85% ± 4%) was similar to the numbers observed in the group that did not receive GSPs (91% ± 4%), as shown in Fig. 2C. However, the number of CPD⁺ cells in the skin samples of IL-12 KO mice that had been treated with dietary GSPs and had received rIL-12 before UVB exposure was only 45% ± 5%, indicating a significant reduction of DNA damage (P < 0.001). These data support the concept that GSPs promote the repair of damaged DNA in UVB-exposed skin through a mechanism that requires IL-12 activity.

If IL-12 is required for GSP-induced DNA repair in UVB-exposed skin, we then asked whether prevention of photocarcinogenesis in mice by dietary GSPs requires IL-12. To test the hypothesis, we examined the effect of dietary GSPs on photocarcinogenesis in IL-12 KO mice. We confirmed that when WT mice were subjected to a standard photocarcinogenesis protocol, dietary GSPs resulted in a significant reduction in UVB-induced skin tumorigenesis in terms of tumor incidence (40%, P < 0.01) and tumor multiplicity (60%, P < 0.001) compared with non–GSP-treated WT mice at the termination of the experiment (Fig. 3A, 3B, left panels). Each group had 20 mice. As shown in Fig. 3C, the growth of tumors was significantly reduced (67%; P < 0.001) in the group of GSP-treated WT mice than the non–GSP-treated mice when measured and expressed in terms of tumor volume/tumor at the termination of the experiment.

In contrast, dietary GSPs did not significantly inhibit photocarcinogenesis in IL-12 KO mice (Fig. 3; right panels). It appeared that the IL-12 KO mice were on a greater risk of UVB radiation-induced skin tumor growth than their WT counterparts, as is evident from the data concerning tumor multiplicity (Fig. 3, right panels). The size of the tumor volume/tumor was higher in the IL-12 KO mice than their WT counterparts (Fig. 3C). These tumor data suggest that prevention of photocarcinogenesis by GSPs was more pronounced in WT mice than IL-12 KO mice.

As we have observed that dietary GSPs enhance the repair of UVB-induced DNA damage in the skin, and it seems that DNA repair by GSPs is IL-12 dependent, the next question was whether GSPs repair DNA damage through the stimulation of NER genes and whether IL-12 is involved in it? For this purpose, shaved backs of the IL-12 KO and their WT counterparts were exposed to UVB (60 mJ/cm²) with and without the treatment of GSPs in diet. Mice were sacrificed 1 hour after UVB exposure, skin samples were collected and epidermal RNA isolated and subjected to the analysis of mRNA expression of some selected NER genes (i.e., XPA, XPC, RPA1, and DDB2) using real-time PCR. The acute exposure of the mouse skin with UVB mildly enhances the levels of NER genes (not significant) compared with non–UVB-exposed skin. As shown in Fig. 4, the mRNA levels of NER genes were significantly enhanced (P < 0.01–0.001) in the skin of WT mice treated with dietary GSPs compared with non–GSPs-treated UVB-exposed WT mouse skin. In contrast, the levels of mRNA expression of XPA, XPC, RPA1, and DDB2 were not significantly elevated in the UVB-exposed skin of IL-12 KO mice, which were given GSPs in the diet. These data suggest the role of IL-12 in the NER gene expression.

As it has been shown that XPA gene plays an indispensable role in the NER pathway (20), we further examined whether XPA gene is required in GSP-mediated DNA repair. For this purpose, XPA-deficient fibroblasts from an XPA-patient and XPA-proficient fibroblasts from a healthy person were exposed to UVB with or without prior treatment with GSPs (20 μg/mL). Cells were harvested either immediately or 24 hours after UVB irradiation and subjected to cytostaining using CPD-specific antibody. CPD⁺ cells were not detectable in non–UVB-irradiated cells whether or not they were treated with GSPs (Fig. 5A). When the cells were analyzed for CPDs immediately after UVB-exposure, no differences were observed in the cells treated with or without GSPs in terms of the number of CPD⁺ cells. This finding suggests that GSPs do not prevent immediate formation of CPDs after UVB exposure and excludes a UVB radiation filtering effect. When the cells were analyzed 24 hours after UVB irradiation, the number of CPD⁺ cells was significantly decreased (79%, P < 0.001) in the XPA-proficient cells (Fig. 5A and B) compared with non–GSPs-treated UVB-irradiated cells, suggesting that GSPs might accelerate the repair of UVB-induced CPDs through an XPA mechanism. A significant reduced number of CPD⁺ cells were also observed in non-GSPs-treated XPA-proficient cells 24 hours after UVB irradiation compared with UVB-irradiated cells immediately after UVB exposure; suggesting that some endogenous defense mechanisms are involved in DNA repair. However, this DNA-repairing effect of GSPs was not observed in the XPA-deficient human fibroblasts 24 hours after UVB irradiation. It may be due to the absence of XPA gene in these cells.

As XPA is a critical component of the NER pathway, we then asked how GSPs affect XPA activity to enhance DNA repair upon UVB irradiation. It has been shown that after UVB irradiation, XPA undergoes translocation from cytoplasm to nucleus, and once inside the nucleus, XPA is a “docking” protein for components of the NER system (27, 28). For this purpose, the shaved C3H/HeN mice were exposed to UVB (60 mJ/cm²) with and without the treatment of dietary GSPs. Mice were sacrificed 1 hour later. Non–UVB-exposed mice served as a control. Cytosolic and nuclear fractions were prepared and subjected to Western blot analysis using XPA-specific antibody. As shown in Fig. 6A, we observed a reduction in the XPA protein level in cytosol after UVB exposure with or without the treatment with GSPs. Furthermore, there was an accumulation of XPA nuclear protein in the UVB-irradiated skin. Dietary GSPs enhanced the accumulation of nuclear XPA protein compared with the non–GSPs-treated UVB-exposed mice. We also determined whether GSPs could enhance the binding of other repair factors to XPA to augment the repair of damaged DNA. Specifically, we assessed the interactions between XPA and ERCC1 upon UVB irradiation with and without treatment with GSPs with XPA immunoprecipitation followed by Western blotting. As shown in Fig. 6B, we found an increase in XPA and ERCC1 association in the GSPs and UVB-irradiated group of mice compared with the UVB-alone group of mice. These data suggest that GSPs promote nuclear translocation of XPA and enhance its interactions with other DNA repair protein, such as ERCC1.

In the present study, we demonstrate a novel mechanism by which GSPs prevent photocarcinogenesis in mice. We show that the dietary GSPs stimulate repair of UVB-induced DNA damage in the form of CPDs in the mouse skin. In addition to the formation of CPDs in UVB-exposed skin, UVA in particular is also responsible for oxidative DNA damage and that can be repaired by a base excision repair mechanism. As UVB-induced CPDs have been recognized as a molecular trigger for the induction of immunosuppression as well as an initiator of skin carcinogenesis, we focused our attention on the repair mechanism of CPDs by dietary GSPs. Moreover, UVB radiations are comparatively more immunosuppressive, mutagenic, and carcinogenic in nature than UVA. The DNA repair process by GSPs is mediated through the induction of IL-12. IL-12 has the ability to repair UVB-induced CPDs (23, 25, 29). The subcutaneous injection of IL-12 at the site of UVB irradiation enhanced the repair of UVB-induced CPDs in GSP-treated IL-12 KO mice. These data support the concept that GSPs stimulate the repair of CPDs from the UVB-exposed skin through a mechanism that involves IL-12 activity. To further prove this concept, we have conducted photocarcinogenesis experiments with dietary GSPs using the IL-12 KO mouse model. The chemopreventive effect of dietary GSPs on photocarcinogenesis was less pronounced in IL-12 KO mice, but a significant inhibitory effect was observed in their WT mice, suggesting that the prevention of photocarcinogenesis by GSPs is mediated through IL-12. The treatment of IL-12 KO mice with GSPs did result in some inhibition of UVB-induced tumor development, although these effects were not statistically significant in these experiments. Our data that suggest a role for GSP-mediated induction of IL-12 in their anticarcinogenic activity are consistent with the demonstrated antitumor activity of IL-12 against cutaneous malignancies and experimental metastases (23, 30–33). As we observed that treatment of WT mice with GSPs increased the levels of IL-12 in UV-exposed mice (26), it is possible that this enhancement of the levels of IL-12 is involved in the inhibition of growth of tumors. IL-12 is a key component of numerous immune functions and has been found to cure or improve the survival of tumor-bearing mice by enhancing in vivo antitumor activity in a number of different tumor models (30–33). The comparison of available information from other tumor models with the present study conducted in the IL-12 KO mouse model indicates that IL-12 has an inhibitory effect on the UV-induced skin tumor development, and its deficiency renders the mice susceptible to photocarcinogenesis.

The NER is a major mechanism of DNA repair in mammalian cells. Since the treatment of GSPs enhances the repair of UVB-induced DNA damage, we further examined whether the repair of UV-induced CPDs by GSPs is mediated via induction of NER genes. Our real-time PCR data reveal that treatment of mice with GSPs increases the levels of NER genes (e.g., XPA, XPC, RPA1, and DDB2) in UVB-exposed skin sites of WT mice compared with non–GSPs-fed mice and that may have contributed in the rapid repair of damaged DNA in the mouse skin. In contrast, this effect of GSPs was not observed in the UVB-exposed skin of IL-12 KO mice. The role of NER was further confirmed by assessing the effect of GSPs on UVB-induced DNA damage in human XPA -deficient cells obtained from human patients suffering from xeroderma pigmentosum disease and XPA-proficient cells from normal healthy persons. Cells derived from patients suffering from xeroderma pigmentosum either lack or have reduced DNA repair capacity due to genetic mutations in several components of the NER. The XPA complementation-type represents the most severe phenotype, because the XPA gene is the most crucial component in the repair process and, thus, cells lacking the XPA gene are completely deficient in NER (29). Our immunostaining data indicated that GSPs were able to repair UV-induced CPDs in XPA-proficient cells but was not able to repair in XPA-deficient cells. These observations indicate that repair of UV-induced DNA damage by GSPs is mediated through the XPA-dependent mechanism. XPA is part of the core incision complex of the NER system (34). Here we also demonstrated that dietary GSPs induces or augments XPA nuclear translocation and then activates the core incision complex in the NER system. Similar observations related with the role of XPA in DNA repair in UVB-exposed skin cells were also found by the treatment of α-melanocyte-stimulating hormone (35). Stoner et al. have studied the effect of diet containing freeze-dried black raspberries on chemical carcinogen N-nitrosomethylbenzylamine–induced dysregulated genes in rat esophagus (36). They have found that 462 of the 2,261 N-nitrosomethylbenzylamine–dysregulated genes were restored to near-normal levels of expression by black raspberries. RNA microarray analysis revealed that dietary black raspberries upregulated the expression of 24 genes whereas downregulated the expression of 12 genes. The upregulated genes were associated with transcription regulation, apoptosis, metabolism, and signal transduction cascades (37). Our study further explains the mechanism that GSPs stimulate nuclear translocation of XPA and binds to other DNA repair protein and thus activates the system. These findings have important implications for the chemoprevention of skin cancer by GSPs and identify a new mechanism by which GSPs prevent UV-induced skin tumor development.

No potential conflicts of interest were disclosed.

Grant Support

This work was financially supported by the Veterans Administration Merit Review Award (S. K. K.).

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