The human squamous carcinoma cell line SNU1 from Seoul National University was grown in tissue culture. SNU cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 ug/ml gentamicin as described previously [3]. Subconfluent monolayers of the cells were detached with 0.25% trypsin in PBS, counted in trypan blue with a hemocytometer, and resuspended in microfuge tubes at 500,000 cells/0.5 ml in RPMI 1640 media without phenol red. These in vitro experiments included hypericin concentrations ranging from 0.1 to 10 μg/mL, dye laser wavelengths of 514, 550, and 593 nm, laser power at the fiberoptic tip of 50, 100, and 150 mW with illumination times of 0–120 seconds for a total light fluence of 0–60 J/cm2 measured at the sample tube liquid interface. After PDT each sample was added to microplate wells at 50,000 cells/0.2 ml in triplicate and incubated for 48 hours at 37°C before cell viability was measured by adding 0.5 mg/ml MTT tetrazolium bromide for 3 hours followed by media aspiration, addition of dimethyl sulfoxide (DMSO) to solubilize the dye product, and optical density of each well recorded at 450 nm with a Molecular Devices microplate reader [3].
In vivo experiments were performed on 3–15 mm diameter tumors growing in athymic nude mice 6–8 weeks after subcutaneous injection of 106 human SCC cells. Tumors were injected with 10 μL of DMSO containing 10 μg hypericin per gm tumor and after 24 hours treated via insertion of a fiberoptic into the tumor center to deliver KTP 532 nm laser light at 500 mW output from a Laserscope operating room medical laser. KTP safety goggles allowed detection of the orange-red dye tumor fluorescence. Hypericin spectra in DMSO had absorbance maxima at 545 and 593 nm and a red-orange fluorescence emission maxima at 594 and 640 nm as shown in Figure 1.
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