Head and neck squamous cell carcinomas (HNSCC) are malignant neoplasms which are classified by its location as it can occur in the mouth (oral cavity), oropharynx, nasal cavity, paranasal sinuses, larynx, nasopharynx and hypopharynx (Genetics Home Reference, 2018). HNSCC is a quite common form of cancer and was highlighted as the sixth most common form of cancer worldwide from an article by Carr et al. 2013, with an incidence of 700,000 cases per year and accounted for 2.8/100,000 cases of all cancers in the United Kingdom in 2011. Head and neck cancers are more than twice as common found among men compared to women and are also more diagnosed among people in their 50s and 60s, although the incidence among younger individuals is increasing. Furthermore, despite improvements in treatment for HNSCC, 5-year survival rates remain relatively uncharged and has hardly improved in the last thirty years (Hattersley et al. 2011).
Risk factors for the development of HNSCC primarily include alcohol and tobacco usage with at least 75% of head and neck cancers caused by this, with a greater risk of developing these cancers being to those people who use both tobacco and alcohol compared to those who use either tobacco or alcohol alone (American Cancer Society, 2017).
Treatment for HNSCC can depend on a number of factors including the exact location of the tumour; the stage of cancer it is currently at as well as the person’s age and general health. Treatment can vary and include: surgery, chemotherapy, radiation therapy, targeted therapy or a combination of these (National Cancer Institute, 2017).
Radiation therapy or radiotherapy is an essential component of treatment for newly diagnosed cancer patients as more than half of these patients will require it (Forker et al. 2015). The process involves depositing high quantities of radiation onto cancer cells which then destroys the cancer. The radiation used includes low and high linear energy transfer (LET) radiation, as described by Baskar et al. 2014, which are used to efficiently kill the tumour cells while minimizing dose to normal tissues to prevent toxicity and damage to these normal healthy cells.
There are three processes for radiation-induced cell death which includes: apoptosis (the normal and controlled death of cells as part of an organism’s development and life cycle), mitotic catastrophe, delayed mitosis-linked cell death which leads to caspase-dependent (apoptotic) or caspase-independent (necrotic) cell death5; and senescence, being the loss of a cell’s power of division and growth (Carr et al. 2013). With HNSCC, radiotherapy promotes all three methods of cell death with the majority of cells undergoing senescence and mitotic catastrophe.
2) Biomarkers of Tumour Radiosensitivity
In this investigation, different sensitive biomarkers were chosen and tested to show how they interact with radiated exposed and unexposed HNSCC cell lines. These biomarkers include: H2AX, M30, VEGF, Bcl-2/BAX as well as a few others.
H2AX is an important cell marker antibody used as a sensitive molecular marker of DNA damage and repair as Mah et al. 2010 described H2AX phosphorylation as a key step in the DNA damage response (DDR), playing a role in signalling and initiating the repair of double-strand breaks (DSBs), which are considered to be among the most lethal forms of DNA damage.
M30 CytoDEATH is a monoclonal antibody, which detects caspase-3 cleaved cytokeratin-18 as a specific tissue marker of apoptosis (Carr et al. 2013). In this investigation M30 was able to detect radiation-induced cell death as well as cells undergoing caspase-dependent death as a consequence of mitotic catastrophe.
Vascular endothelial growth factor (VEGF) has been identified as an important independent prognostic factor for HNSCC, according to Gong et al 2016. VEGF includes important signalling proteins involved in both angiogenesis and vasculogenesis. Angiogenesis being the formation of new blood vessels from pre-existing vessels; is essential for tumour growth and metastasis, which means that it is an important target for anticancer treatment. Thus, increased production of VEGF leads to increased angiogenic activity and tumour progression.
BAX (Bcl-2-associated X protein) has been described in a study by Budworth et al. (2012) as an apoptosis biomarker of radiation exposure. They identified that, at the protein level, BAX was elevated after radiation exposure and was used in the investigation as a surrogate indicator for the role of apoptosis. They also recorded significant increases of BAX proteins at 6 and 24 hrs after both 2 and 6 Gy exposures (p,0.05), thus confirming it as a useful sensitive marker of radiation exposure.
3) Aims of the Study
The aims of this study were to lyse cell pellets from both HNSCC cell lines with and without irradiation to extract the protein. This protein was then quantified and then a set amount was loaded onto a polyacrylamide gel for separation. Western blotting techniques were then used to transfer the proteins onto polyvinylidene difluoride (PVDF) membranes which were then probed with specific antibodies to determine if there were a difference between irradiated and non-irradiated samples. These specific antibodies included H2AX (DNA damage response), M30 (apoptosis), VEGF (angiogenesis), Bcl2 and BAX (apoptotic-related proteins). The aim is to identify some specific proteins which are changed following irradiation so that these can then be used to screen the tissue for a response. These identified differences between the two samples may then be used for future studies with the ultimate aim being to predict a response of a tumour to irradiation ex vivo before the patient is exposed to the treatment.
4) Ethical Implications
Generally, if cells extracted from a patient are wanted to be used for research purposes then consent from both the patient whose cells they belong to and the hospital in which the patient is staying in is required. However, for this project an ethical consent form is not required as the patient has given consent and the cell pellets can be bought online from an online biorepository such as the American Type Culture Collection (ATCC), which collects, stores, and distributes standard reference microorganisms, cell lines and other materials for research and development. In addition, the cells used are immortalised, meaning that they do not die after a set number of cell divisions, and irradiated. The cells used in this project were provided by the University of Michigan, USA (UMSCC – University of Michigan Squamous Cell Carcinoma).