OBJECTIVE: Optical spectroscopic tools exist that allow open surgical and minimally invasive assays of intrinsic tissue optics. Optical detection of cellular and tissue viability may offer a minimally invasive way to assess tumor responsiveness to chemotherapies. We report on an optical spectroscopic change that precedes apoptotic cell death and appears related to NAD(P)H autofluorescence.
METHODS: The cell lines SW 480 and U87-MG were grown in culture and treated with cisplatin 100 μg/ml and tamoxifen 10 μM, respectively. Fluorescence spectroscopy at 355 nm excitation and 460 nm emission were collected. MTS assays were used to determine cell viability. Cell lysates were analyzed for NAD(P)H concentrations by mass spectroscopy. RESULTS: Autoflourescence at 355 nm excitation and 460 nm emission declines markedly despite normalization for cell number and total protein concentration after treatment with tamoxifen or cisplatin. The autofluorescence drop precedes the loss of cell viability as measured by MTS assay. For example, the relative viability of the U87-MG cell treated with tamoxifen at hours 0, 8, 12 and 24 of treatment was 100 ± 6, 85 ± 6, 53 ± 9 and 0 ± 3. The relative fluorescence at the same time points were 100 ± 2, 57 ± 6, 47 ± 3, and 0 ± 1. TUNNEL assays confirm that cell death is via apoptosis. The key cellular fluorophore at these wavelengths is NAD(P)H. Mass spectroscopic analysis of cell lysates at these time points reveals a drop in NAD(P)H concentrations that is parallel to the loss of fluorescence signal. CONCLUSIONS: NAD(P)H autofluoresence decline precedes apoptotic cell death. This may allow the design of minimally invasive spectroscopic tools to monitor chemotherapeutic response.