The Preventive Effects Of Two Marine Anti-Oxidant Peptides Against UVB Induced Mouse Skin Cancer And Exploration Of Potential Mechanism

Aim: The aim of this study was to establish chronic day-light mimicking UVB(180m J/cm2) induced skin carcinogenesis model in Kunming hairless mice and to investigate the preventive effects of marine antioxidant peptides: polypeptides from chlamys farreri(PCF) and collagen peptides from walleye pollock skin(CPWPS)against UVB induced skin carcinogenesis, focusing on AQP3, AQP9, EGFR and AKT as potential mechanisms. Methods: CPWPS cytotoxicity was screened with CCK-8method in Ha Ca T cells(50, 100, 200, 300, 500, 1000, 2000 mg·L-1), non-toxic doses were selected for subsequent experiments, in which the inhibitatory effect of PCF and CPWPS on skin squamous-cell carcinoma cell line A431 were assessed with CCK-8assay. Additionally, in vivo work randomly assigned Kunming hairless mice into different groups: control group, UVB model group, 5% vitamin C(VC) group, 5%PCF group, 5% CPWPS group and 20% CPWPS group. All the animals except for those in control group received 180 m J/cm2 UVB exposure three times a week for a total of 25 weeks to induce skin carcinogenesis. Pathological changes in skin after 5,10, 15, 20 or 25 week UVB exposure were assessed with visual observation and Hematoxylin and Eosin(HE) staining. Immunohistochemistry was utilized to assess the expression levels of PCNA in control and UVB model group, as well as the expression levels of AQP3, AQP9, EGFR and p-AKT in all the groups after 25 week treatment. Results: In vitro work revealed significant cytotoxicity of CPWPS only at concentrations higher than 300 mg·L-1(P<0.05). However, 1.42, 2.48, 5.68 mmol.L-1PCF and 50, 100 or 200 mg·L-1 CPWPS treatment in A431 cell did not remarkably inhibit cell proliferation(P>0.05). On the other hand, our in vivo work demonstrated directly observable rythema and scale on the hairless mice skin following five week chronic UVB exposure; 10 week exposure led to scale and linting;pin-point sized papula started to emerge from week 15; more papula were observed by week 20, and tumor formation was confirmed by week 25, these symptoms were alleviated to varied degrees in mice treated with marine antioxidant peptides at the same time. The tumor formation in all the three groups of mice treated with marine antioxidant peptides were delayed relative to model group mice, with less tumorformation on the back per mice. Notably, only 1.80±0.45 tumor on average were observed on the back per mice treated with 20% CPWPS by week 25(P<0.05). Under optic microscope, HE stained sections indicated no difference between Kunming hairless mice and normal Kunming mice, except for thinner epidermis, less follicles and sudoriferous. After five week UVB exposure, thicker epidermis was observed in the UVB model group hairless mice samples comparing to control; 10 week UVB exposure led to hyperkeratosis of epidermis and irregular trochanterellus that slightly stretched downward; more prominent hyperkeratosis was present after 15 week UVB exposure and nest of cancer started to emerge. After 20 week UVB exposure,hyperkeratosis became more obvious, more nest of cancer were present with slight deposit of melanin and increased nucleus/cytoplasm ratio. Infiltration and breaching of basal layer were observed. Irregular cell alignment and dysplasia were also noticeable. Finally, 25 week UVB exposure resulted in extensive hyperkeratosis,diffuse dysplasia and more prominent nests of cancer along with horn pearls.Immunohistochemistry revealed higher PCNA protein expression in UVB model group relative to control group(P<0.05). These data indicated successful establishment of skin cancer model in Kunming hairless mice. Meanwhile, these symptoms were alleviated to varied degrees in mice treated with marine antioxidant peptides at the same time. Moreover, immunohistochemistry demonstrated remarkably higher expression levels of AQP3, AQP9, EGFR and p-AKT in UVB model group comparing to control group(P<0.05), while 5% PCF, 5% CPWPS, 20%CPWPS and 5% VC treatment all resulted in significantly lower expression levels of AQP3, AQP9, EGR and p-AKT(P<0.05). Among different treatments, 20% CPWPS exhibited the most prominent effect reducing the expression levels(P<0.05).Conclusions: For the first time, involvement of EGFR, AKT, AQP3 and AQP9 in UVB induced skin carcinogenesis and tumor progression was revealed. Topical application of marine antioxidant peptides(PCF and CPWPS) exerted preventive effects against 180 m J/cm2 UVB induced skin carcinogenesis, in which 20% CPWPS had most remarkable effect. The underlying mechanism of these peptides is associated with inhibitory effects on the expression of EGFR, AKT, AQP3 and AQP9.