Psoralen as a Photosensitizers for Photodynamic Therapy by Means of In Vitro Cherenkov Light

Abstract

Possible enhancements of DNA damage with light of different wavelengths and ionizing radiation (Rhenium-188-a high energy beta emitter (Re-188)) on plasmid DNA and FaDu cells via psoralen were investigated. The biophysical experimental setup could also be used to investigate additional DNA damage due to photodynamic effects, resulting from Cherenkov light. Conformational changes of plasmid DNA due to DNA damage were detected and quantified by gel electrophoresis and fluorescent staining. The clonogene survival of the FaDu cells was analyzed with colony formation assays. Dimethyl sulfoxide was chosen as a chemical modulator, and Re-188 was used to evaluate the radiotoxicity and light (UVC: λ = 254 nm and UVA: λ = 366 nm) to determine the phototoxicity. Psoralen did not show chemotoxic effects on the plasmid DNA or FaDu cells. After additional treatment with light (only 366 nm-not seen with 254 nm), a concentration-dependent increase in single strand breaks (SSBs) was visible, resulting in a decrease in the survival fraction due to the photochemical activation of psoralen. Whilst UVC light was phototoxic, UVA light did not conclude in DNA strand breaks. Re-188 showed typical radiotoxic effects with SSBs, double strand breaks, and an overall reduced cell survival for both the plasmid DNA and FaDu cells. While psoralen and UVA light showed an increased toxicity on plasmid DNA and human cancer cells, Re-188, in combination with psoralen, did not provoke additional DNA damage via Cherenkov light.

Keywords: Cherenkov light; FaDu cells; Re-188; plasmid DNA; psoralen.

Figure 1

Fluorescent measurement of an agarose gel for combining psoralen with UV-366 nm. The aperture detects the intensity spectra (bottom) of the fluorescent gels and displays them visually (top). The figure is inverted, thus showing a dark color for high intensities. Since this measurement is not quantitative, the intensity scale only shows arbitrary units. Markers are placed in lanes 1, 11, and 20. Lane 2 and 18 show untreated plasmid DNA, whilst lane 19 contains enzymatically linearized plasmid as the control. In lanes 4–10 and 12–17, different psoralen concentrations (0, 0.1, 1, 10, 25, 50, 75, 100, 200, 400, 500, 700, 900 µM) were irradiated with UV-366 nm for 90 min. Each lane was normalized and shows the running behavior of the different setups. A higher concentration of psoralen showed a higher amount of open circular conformations (OCs). Lane 3 demonstrates non-UV-activated psoralen (900 µM).

Figure 2

Exemplary concentration–effect relations for the combination of psoralen and varying irradiation times of UV-366 nm for the SC conformation. The longer the irradiation with UV-366 nm, the more intense the fractions of the open circular confirmation as a consequence of the SC conformation quickly dropping to zero. The error bars show the SEM.

Figure 3

UV irradiation time–effect relations for the combination of psoralen with UV-254 nm. (a) OC fraction as a function of irradiation time, (b) OC fraction as a function of psoralen concentration. The effects are displayed as changes in the migration distances of DNA in relation to the markers bp. To increase the irradiation time, the effect as in relative fluorescence intensity also increased. However, this effect was nearly concentration independent. The error bars show the SEM.

Figure 4

Fluorescent measurement of an agarose gel for combining psoralen with UV-366 nm with an additional 0.2 M DMSO for several lanes. The figure is inverted, thus showing a dark color for high intensities. Markers are placed in lanes 1, 11, and 20. Lane 2 and 18 show the untreated plasmid, whilst lane 19 contains the linear plasmid. In lanes 4–10 and 12–17, different psoralen concentrations (0, 1, 10, 100, 200, 400, 750 µM) were irradiated with UV-366 nm for 30 min, where an additional 0.2 M DMSO was present in lanes 12–18. Each lane was normalized and shows the running behavior of the different setups. In comparison to Figure 1, the additional DMSO did not produce a different behavior. Lane 3 demonstrates non-UV-activated psoralen (900 µM).

Figure 5

Light intensity of different amounts of Re-188 in a 24-well plate. The picture is inverted, thus, showing increasing intensities of light from left to right (f.l.t.r.: 0, 4.125, 8.25, 16.5, and 33 MBq). The spectrum (bottom) was detected by the camera and is shown visually at the top of the figure. If more Re-188 radioactivity is present, the larger the light intensity will be. All intensities were not quantifiable.

Figure 6

Light intensity of different setups of Re-188 and DMSO as preliminary investigations. The spectra show the detected light intensity in arbitrary units, thus, they are not quantifiable. (a) The first well did not contain radioactivity (2000 µL distilled water) and did not consequently show light intensity. The other wells contained a nearly constant amount of Re-188 (approximately 6.85 MBq) and varying volumes of water (f.l.t.r. 200, 500, 800, and 1000 µL). More light intensity was seen for larger water volumes. The spectrum showed that there was no linear behavior between the volume of the medium and the light intensity. However, a saturation was reached for a volume large enough to produce a 1 cm water column (depends on size of well). Measured light intensity: 7 Mio (background, no activity), 22 Mio, 27 Mio, 28 Mio, 29 Mio integral light volume intensity in arbitrary units for each well. (b) All wells contained a constant amount of Re-188 (approximately 6.7 MBq), varying amounts of DMSO, and additional water to reach a constant volume of 2000 µL. From left to right, an increasing amount of DMSO was added to the wells (0, 10, 100, 500, and 1000 µM). The same light intensity was seen for every DMSO concentration, since it is not photo-activated by Re-188. Measured light intensity f.l.t.r.: 28 Mio, 26 Mio, 26 Mio, 27 Mio, 27 Mio integral light volume intensity in arbitrary units for each well.

Figure 7

Dose–effect relation for Re-188 after 24 h incubation. The mere radiotoxicity of Re-188 had an influence on all conformations. Supercoiled (SC) transformed into open circular (OC) and eventually into linear (L) conformations for higher doses. The error bars show the SEM.

Figure 8

Exemplary dose–effect relation for Re-188 in combination with psoralen after 24 h incubation demonstrated an increasing fraction of open circular plasmid DNA. The influence of DMSO was tested. Psoralen produced a small additional effect, as visible in the difference between the continuous and dashed line. The error bars show the SEM.

Figure 9

Dose–effect relation on plasmid DNA for Re-188 in combination with psoralen and with (a)/without (b) a 60-min UV-366 nm irradiation. (a) shows the additional fluorescence intensity of open circular conformation for mere radiotoxicity with/without UV-366 nm irradiation. There was a difference between the psoralen and the DMSO equivalent effect. In (b) the difference of the fluorescence intensity is shown by comparing UV-366 nm irradiation and no irradiation. The effect was larger with UV-366 nm irradiation. The error bars show the SEM.

Figure 10

Concentration–effect relation for a combination of psoralen and UV-366 nm (after 24 h preincubation of psoralen in FaDu cells). The longer the irradiation with UV-366 nm, the lower the cell survival fraction (SF). This effect was enhanced by adding psoralen. Without irradiation, there was no difference in survival for any psoralen concentration. The error bars show the SD_pooled.

Figure 11

Concentration–effect relation for a combination of psoralen and Re-188 on the survival (SF) of FaDu cells (mere radiotoxicity is additionally shown). No significant difference between mere radiotoxicity and adding of psoralen was visible. The error bars show the SD_pooled.