Patrick Badger and Dr. Jason Hansen, Department of Physiology and Developmental Biology
Ultraviolet light (UV) plays a critical role in the etiology of skin cancer, contributing to over 95% of both melanoma and non-melanoma diagnoses. It consists of several wavelengths, among which UVB is the most harmful, causing inflammation associated with sunburn and constituting the principal cause of skin cancer-related death.
This study investigates a molecular cascade potentially responsible for carcinogenesis in human skin incident to UVB exposure. That cascade begins when energy from UVB radiation induces in skin cells what is known as a state of oxidative stress; oxidative stress occurs in all cell types and may be caused by nearly limitless sources of physical and chemical cellular damage. In the case of UVB, high energy radiation provides induces the movement of electrons to and from chemical species that occur naturally in cells, generating what are known collectively as reactive oxygen species (ROS). In excess, these reactive species may cause a wide array of damage, modifying DNA and cellular proteins such that cells either die or exhibit a range of aberrant functions.
In time, both acute oxidative stress (such as that experienced after intense sunburn) and chronic oxidative stress (such as that caused by a lifetime of sun exposure) can lead to tissue-level changes, the most dangerous of which is cancer. Because oxidative stress is a known consequence of UVB exposure, it is reasonable to suggest that it might contribute to some or all of the processes that result in the development of skin cancer. However, due to the complexity of the cellular oxidative stress response, it has heretofore been difficult to identify which molecular pathways are actually at play.
This study expands understanding of the mechanisms of oxidative stress contributing the development of skin cancer following UVB exposure by identifying the specific molecule responsible for mediating the cellular oxidative stress response in that context. It also excludes the most likely other candidate by showing that it does not respond to oxidative stress in a significant way under such conditions.
This study relied mainly on two techniques: native polyacrylamide gel electrophoresis (native PAGE) and high-performance liquid chromatography (HPLC).
Native PAGE entails the separation of protein mixtures drawn through a porous gel by electric current on the basis of overall molecular charge; specific proteins are then tagged with fluorescent markers and imaged on a scanner that can detect the fluorescence. The relative concentrations of proteins under different cellular conditions can be observed in this way (i.e. the darker the fluorescent image, the higher the protein concentration in that image). HPLC is also used to determine relative concentrations, but does not require imaging. Rather, it takes advantage of the fact that certain molecules are more soluble in some liquid solvents than they are in others; by using several different solvents to run protein mixtures through a narrow tube, it can separate specific proteins from a mixture, using one solvent to move one molecule through the tube by washing it from the mixture, and so on.
Briefly, this study utilized native PAGE as described to determine the relative concentrations of a molecule known as Thioredoxin (the most likely alternative oxidative stress resolving molecule to glutathione) in human keratinocytes treated with both UVB and hydrogen peroxide (a positive control to show what a highly oxidized sample would look like for comparison), as well as untreated cells (a negative control to show what relatively un-oxidized samples should look like.
Similarly, HPLC was used as described to determine glutathione concentrations in samples prepared under the same set of conditions used to prepare those analyzed with native PAGE.
PAGE data from many independent experiments indicate that Thioredoxin concentrations did not change in UVB treated cells compared to untreated cells, indicating that it does not increase in concentration following UVB exposure, and therefore does not play a significant role in resolving UVB-induced oxidative stress.
Likewise HPLC data demonstrate that glutathione concentrations increase significantly following UVB exposure, indicating it is primarily responsible for mediating how human skin cells respond to UVB-induced oxidative stress, doing the lion’s share of oxidative damage control in that context.
These results are significant because specific molecules mediate the resolution of oxidative stress resulting from specific cellular insults (i.e. one molecule might handle oxidative stress caused by bee sting while another might resolve oxidative stress caused by blunt force trauma). Knowing which molecule is paired with which kind of insult facilitates the identification of the specific cellular processes initiated by that stress, which can then lead to the identification of molecular mechanisms responsible for outcomes such as cancer or other pathologies.
This study identifies glutathione as the main molecule responsible for resolving oxidative stress in UVB-irradiated human keratinocytes, thereby elucidating the initiator of a cascade of events likely responsible for the development of skin cancer. While further research is required to identify all of the relevant mechanisms the follow glutathione’s remedial activity, this result signifies significant progress.