We hypothesize that the chemopreventive agents 4-HPR and progesterone induce apoptosis in some susceptible ovarian surface epithelial cells and inhibit growth in other less sensitive epithelial cells and that these are the mechanisms by which they prevent ovarian cancer. We further hypothesize that these mechanisms are mediated through the mitochondria by altering membrane permeability transition and inducing reactive oxygen species, decreasing NADH and increasing redox (FAD/(FAD+NADH), and inducing the activation of caspase 9 and later caspase 3 prior to the induction of apoptosis and/or growth inhibition. Cancer cells are thought to behave in the opposite manner with an increase of NADH and a decrease in redox, as they grow at an unrestricted pace. We hypothesize that these changes can be evaluated by fluorescence spectroscopy of endogenous fluorophores. Therefore it can serve as a biomarker for drug response and molecular activity within the target cells. . This will allow the development of minimally invasive techniques to differentiate the activated, and potentially early neoplastic cells from the quiescent cells on the ovarian surface and subsurface as a diagnostic tool for high risk women who are being screened for ovarian cancer. Additionally this technology can be used as a marker for chemopreventive drug activity in women on preventive agents due to their risk of ovarian cancer.

Preliminary Results

We investigated the use of fluorescence spectroscopy as a biomarker for drug activity in eighteen adult female Rhesus macaques given fenretinide (4-HPR), oral contraceptives (OCP), the combination (4-HPR + OCP), or no medication for 3 months (6,11). Exploratory laparotomy was done pre- and post-drug to assess intermediary biomarkers of neoplastic phenotype, proliferation, response pathways, growth-regulatory, and metabolic markers. Fluorescence emission spectra were plotted for each group pre- and post-drug, means were overlaid on these plots, and normalized. Fluorescence intensities were compared using the 2-tailed Student's t- test, (p, 0.1-0.01) (Fig. 8). Histochemical markers showed no significant trend, due both to loss of surface epithelium and small numbers. However, fluorescence spectroscopy showed increased intensity at 450 nm excitation, 550 nm emission, correlating with increased FAD presence. The 4-HPR group (p = 0.01) showed higher intensity than the OCP group (p = 0.05-0.07) when compared to the controls. Decreased emission was seen at 350 nm excitation, 450 nm emission correlating with decreased NAD(P)H presence. The OCP group showed the largest change (p <0.01) consistent with decreased NAD(P)H presence and the control group the smallest change. Fluorescence spectroscopy was the most sensitive marker for drug activity and the apparent increase in NAD and FAD in the 4-HPR group is consistent with the effect of 4-HPR observed in cell culture (Fig. 8). The differences between the OCP and the 4-HPR group suggest a different mechanism of activity of these drugs.

FAD related change	NADH related change
Graphical representation of the FAD an the left and the NADH on the right. Although variances are large, there are still significant trends between the drug groups, suggesting that fluorescence spectroscopy has the capability of identifying a specific effect of the drugs on the ovary.