Primarily localized in the cytosol, human NAD(P)H: quinone oxidoreductase isozyme-1 (hNQO1) is a homodimeric flavoprotein capable of catalyzing the two-electron reduction of quinones to hydroquinones in the presence of cofactor NAD(P)H. Due to its antioxidant abilities, hNQO1 has been found to be a key protective agent in the cell. However, its impact has been found to be favorable to both healthy cells, and cells derived from different cancerous tumor tissues. The latter has made hNQO1 a suitable target for therapeutic, surgical, and prevention practices. It has been previously reported that in two-dimensional cultures, hNQO1 activity is higher in the post confluent growth phase than in the exponential growth phase. Herein, hNQO1 activity and expression in several ovarian cancer cell lines (OVCAR-5, OVCAR-8, and SHIN-3) was evaluated in two-dimensional monolayers during the exponential and post confluent growth. Immunocytochemical analysis was conducted on to determine whether hNQO1 localization changed within the studied cell monolayers as they matured. Protein quantification assays indicated that there was a significant increase in total protein content as cells matured. To determine hNQO1 activity, an ultraviolet-visible (uv-vis) spectrometric method was used to observe the reduction of a standard hNQO1 substrate, 2,6-dichlorophenolindophenol (DCPIP), in the presence and absence of dicoumarol, a known hNQO1 inhibitor. The results of the activity study revealed an increase in hNQO1 activity during the first five days of growth for each of the studied cell lines with the fifth day displaying the highest activity, followed by a decline between the fifth and seventh days. To further confirm the presence and activeness of hNQO1 within each of the studied cell lines, an hNQO1 activated fluorescent probe developed in the McCarley group was employed for imaging via confocal microscopy.

Evaluation of human NAD(P)H:Quinone Oxidoreductase Isozyme-1 (HNQO1) expression and activity in several human ovarian carcinoma cell monolayers

253rd National Meeting (2017)

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