| Purpose1. To establish a mouse model of skin cancer induced by UV radiation. It would be significant for the further study on the prevention and treatment of skin cancer.2. To establish some parameters of ALA-PDT for SKH-1 hairless mice, such as the optimal illumination time, ALA concentration and red light radiation dose.3. To explore the preventive effect of weekly ALA-PDT on ultraviolet (UV) radiation induced SKH-1 hairless mice with squamous cell carcinoma (SCC).4. To investigate the mechanism of the prevention effect of ALA-PDT and a relationship between the delayed skin cancer and mitochondrial pathway of apoptosis.Methods1. Female SKH-1 hairless mice were given 28 weeks of ultraviolet radiation in order to establish skin cancer models. Tumor growth and the histopathological changes were observed and recorded.2. To get the optimal ALA incubation time, fluorophotometer was employed to measure the intensity of PpIX at different time poin after 5% ALA applied. To get the optimal ALA concentration and red light dose, the SKH-1 hairless mice were divided into three groups with 2%, 5% and 10% ALA concentration respectively. The light doses of 0.6J/cm2,1.2 J/cm2,1.8 J/cm2 and 2.4 J/cm2 were applied to four back reigns after 3 hours of ALA incubation. 3. Female SKH-1 mice (n = 100) were randomly divided into the UV (n = 30) group, UV / ALA-PDT group (n = 30), ALA-PDT group (n = 30) and control group (n = 10), i.e. A, B, C, D group. Weighing, measuring skin fold thickness were taken once every 4 weeks and the cancer occurrence were observed after10 weeks. The tumor size and numbers were recorded until 28 weeks. 4. Western blot technique was employed to determine the release of cytochrome c and the expression of caspase-9/3 before and after ALA-PDT.Results1. Erythema and scaling were observed after 1 week of UV irradiation. After 8 weeks of UV exposure the skin became thicken. After 12 weeks of continuous irradiation, needle size papules started to appear, and gradually increased just like a cauliflower. The pathological features of all the papules and tumors were SCC.2. The peak of PpIX fluorescence was on the time point of 3 hour. No significant difference was found between the different concentrations of ALA. The skin damage would be more serious just as the dose of red light increased. The optimal dose of red light was 1.2J/cm2.3. The weight of mice were significantly lower in group A, B than that in group C, D (P <0.05), and the skinfold thickness were much thicker than that in group C, D (P <0.05). The first eruption normally was found at the 12th week, and the histology presented as AK in group A. However, it occured 2 weeks later in group B. The median time of tumor-free survival was the 24th week in group A and the 21th week in group B, the difference was significant (P <0.05). The average number of papules per mouse in group B was less than the group A, but there was no significant difference in the average tumor volume between two groups (P> 0.05).4. Semi-quantitative comparison of the results from Western blot, we found the release of cytochrome c and the expression of caspase-9 and caspase-3 and their activatied fragments were significantly increased after ALA-PDT was applied and the peak time was 3 hours after the treatment.Conclusions1. Long-term solar UV radiation can create a stable SKH-1 hairless mouse skin squamous cell carcinoma (SCC) model, the observation and histological analysis further showed how papules developed into invasive SCC.2. 3 h, 2% ALA cream, 1.2J/cm2 were the optimal ALA incubation time, the best drug concentration and best red light dose for the use of ALA-PDT in the prevention study.3. This study confirmed that weekly ALA-PDT significantly delayed the emergence of skin cancer, reduced the number of tumors, but no significant inhibition effect on tumor volume.4. ALA-PDT induced apoptosis of SKH-1 hairless mouse tumor cells by the mitochondrial pathway. This may be the mechanism of ALA-PDT to prevent squamous cell skin cancer in SKH-1 hairless mouse.
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