DOI: https://doi.org/10.15368/theses.2014.187
Available at: https://digitalcommons.calpoly.edu/theses/1340
Date of Award
12-2014
Degree Name
MS in Biomedical Engineering
Department/Program
Biomedical and General Engineering
Advisor
Lily Laiho
Abstract
Skin cancer is the most common type of cancer in the US. The American Academy of Dermatology estimated that more than 3.5 million new cases of skin cancer are diagnosed in the US each year and 1 in 5 Americans will likely to develop skin cancer in their life time. Most cases of skin cancer are caused by exposure to ultraviolet (UV) radiation from the sun. Some of the most common sunscreen ingredients are unstable and can form harmful radicals upon exposure to UV radiation. There is a strong clinical need for a more stable and effective sunscreen ingredient such as bovine milk phospholipids (MP). Phospholipids were shown to have beneficial health effects such as regulation of the inflammatory reactions, protective effects against colon cancer, and reducing cardiovascular risk factors. Previous histology and MTT tissue viability research studies suggested that MP act upon skin cells in a protective manner against UV radiation.
This thesis aims to further investigate the protective effects of bovine milk phospholipids by evaluating the expression of a UV-induced DNA damage marker, cyclin-dependent kinase inhibitor, p21WAF1/CIP1. Western blots were used to quantify p21 expression in human keratinocytes in four categories of samples: No-UV, UV, UV+MP, MP and in HeLa (p21 positive control). In the No-UV samples, cells were not irradiated by UV light. However, in the UV samples, keratinocytes were exposed to a UV dosage of 10 mJ/cm2. In the UV+MP samples, keratinocytes were first treated with 1% MP solution (w/v) in their culture media for 24 hours then exposed to a UV dosage of 10 mJ/cm2. In MP, keratinocytes were treated with 1% MP solution in their culture media for 24 hours. Total cell proteins were extracted 24 hours post-UV irradiation. The same amount of protein from each sample (determined by BCA assay) was loaded into a 4-12% Bis-Tris SDS-PAGE gel, run under denaturing, non-reducing conditions then blotted and treated with antibodies for the quantitative detection of p21 proteins. Finally, intensities of p21 protein bands were analyzed by using ImageJ software.
Under non-reducing conditions, three p21 proteins covalently bonded with each other showed up as 63 KDa molecules on the PVDF membrane. The UV, and HeLa samples showed a 2.28 fold, and 1.23 fold increase in p21 expression, respectively, compared with the No-UV samples control. The MP samples showed a 0.948 fold decrease in p21 compared with the No-UV samples, and the UV+MP samples showed only a 1.13 fold increase in p21. When comparing with the UV sample, the UV+MP sample has 50.4% less p21 expression. Less p21 expression in the UV+MP sample compare with the UV sample suggested that less DNA damage occurred in the sample that was treated with milk phospholipids. This result strongly suggests that 1% bovine milk phospholipids can protect skin cells from UV induced DNA damage.