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theses

Melanoma is the most aggressive form of skin cancer; in 2018 about 90,000 new cases and 9,000 deaths are expected in the United States. Our group described the oncogenic potential of a normal neuronal cell receptor, metabotropic glutamate receptor 1 (mGluR1), when aberrantly expressed in melanocytes, the pigment producing cells. Deregulated melanocytic cell proliferation leads to neoplastic transformation and progression to spontaneous metastatic melanoma in a transgenic mouse model. The natural ligand of mGluR1 is glutamate and it is well known that all cells, particularly cancer cells, depend on glutamine/glutamate for growth. We showed that mGluR1 expressing cells establish autocrine/paracrine loops by secreting glutamate to the extracellular space to ensure constitutive activation of the receptor, mGluR1 and promote cell growth. Treatment of these cells with pharmacological inhibitor of mGluR1 or through genetic manipulation by silencing RNA to reduce the receptor expression, render the receptor nonfunctional and led to cell cycle arrest at the G2/M phase followed by apoptotic cell death. Riluzole is FDA approved for the treatment of amyotrophic lateral sclerosis (ALS) and one of its functions is the inhibition of glutamate release. This allows the drug to function as an antagonist to mGluR1 activity. Examination of riluzole treated melanoma cells reveals elevated levels of phosphorylated histone H2AX (γH2AX), a protein marker for DNA double-stranded breaks (DSBs). Furthermore, increased ROS levels and decreased intracellular glutathione were also detected in riluzole-treated melanoma cells. We hypothesize that riluzole interacts with the glutamate/cystine antiporter (xCT) to reduce glutamate efflux and cystine influx. Cystine, when reduced to cysteine, is a critical component in glutathione synthesis. Limiting the influx of cystine reduces glutathione levels in the cell, resulting in oxidative DNA damage. The overall goal of this thesis is to determine the consequences of riluzole-induced DNA damage when an ROS scavenger such as N-Acetylcysteine (NAC) is included in the growth media. Effects on DNA damage was assessed using protein markers for single-stranded and double-stranded DNA breaks (SSBs). Alkaline and neutral conditioned COMET assays are used to ascertain the types of riluzole-induced DNA damage. Lastly, by using flow cytometric intracellular staining analysis and guided by the presence of inhibitors to two different DNA repair pathways we were able to conclude that the oxidative damage induced by riluzole is likely DSBs and at Non Homologous End Joining (NHEJ) repair pathway is the preferred double strand break pathway used by mGluR1-expressing melanoma cells.

ETD_9205

doi:10.7282/T3MP56X2

M.S.

Includes bibliographical references

by Robert M. Cerchio Jr.

ETD graduate

The author owns the copyright to this work.