Determining the effect of glutaredoxin-1 overexpression on the growth of B16F0 tumors in mus musculus
Johnson, Ryan David Patrick
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BACKGROUND: While advances in medicine have led to increased survival rates, cancer is responsible for millions of deaths each year. Melanoma itself accounts for tens of thousands of these deaths, and is capable of progressing rapidly. If left untreated, patient prognosis is bad and survival is unlikely. With distant metastasis, survival rates past five years are under ten percent. The link between angiogenesis and tumor growth has been well established, and therefore anti-angiogenic factors should show a decrease or slowing of cancer growth. Glutaredoxin-1 (Glrx), an enzyme residing in the cytosol, is responsible for removing glutathione (GSH) from GSH-protein adducts. This plays a role in reversible cellular signaling. It has been shown that increased levels of global and endothelial cell (EC) specific Glrx leads to decreased levels of revascularization following hind limb ischemia in mice. Few studies however have compared the overexpression of Glrx to melanoma tumor growth. OBJECTIVE: To examine how Glrx overexpression in endothelial cells (EC) influences B16F0 murine melanoma cell growth in C57BL6 mice. METHODS: Eight Glrx overexpressing double transgenic (DTG) and seventeen control mice (Ve-cadherin or WT) between the ages of 3 and 4 months old each received subcutaneous injection with B16F0 mouse melanoma cells. After 2 weeks, the mice were euthanized to remove tumors. Weight and size of tumor were recorded. Tumor, lung, and tail samples were taken. Real-time polymerase chain reaction (qPCR) of tumor samples were performed. Genotypes were confirmed via western blot of lung and PCR of tail DNA. Excised tumor tissue was in part fixed to examine for angiogenesis and macrophage markers. VEGF ELISA was performed on tumor samples. Immunohistochemistry (IHC) testing for Isolectin B4 and CD68 was performed on frozen sections. RESULTS: Slightly higher average tumor weight was found in DTG mice (m=1229.45 mg ± 202.89 mg, n=8) when compared to the control (m=827.32 mg ±172.62 mg, n=17). Lower gene expression of VCAM, VEGFA, IL-6, IL-1B, Kdr, and HIF1 was found in the DTG mice when compared to the control. The expression of CD68 was significantly lower (P<0.05). VEGF ELISA showed higher concentrations of VEGF per total protein in the DTG mice versus the control mice. IHC showed DTG had higher endothelial cell signaling by Isolectin B4, and higher macrophage signaling by CD68. CONCLUSIONS: The growth of B16F0 murine melanoma is not suppressed by the overexpression of Glrx in EC of C57BL6 mice.