Light-Enhanced Cytotoxicity of Doxorubicin by Photoactivation

Abstract

The combination of photodynamic therapy with chemotherapy (photochemotherapy, PCT) can lead to additive or synergistic antitumor effects. Usually, two different molecules, a photosensitizer (PS) and a chemotherapeutic drug are used in PCT. Doxorubicin is one of the most successful chemotherapy drugs. Despite its high efficacy, two factors limit its clinical use: severe side effects and the development of chemoresistance. Doxorubicin is a chromophore, able to absorb light in the visible range, making it a potential PS. Here, we exploited the intrinsic photosensitizing properties of doxorubicin to enhance its anticancer activity in leukemia, breast, and epidermoid carcinoma cells, upon irradiation. Light can selectively trigger the local generation of reactive oxygen species (ROS), following photophysical pathways. Doxorubicin showed a concentration-dependent ability to generate peroxides and singlet oxygen upon irradiation. The underlying mechanisms leading to the increase in its cytotoxic activity were intracellular ROS generation and the induction of necrotic cell death. The nuclear localization of doxorubicin represents an added value for its use as a PS. The use of doxorubicin in PCT, simultaneously acting as a chemotherapeutic agent and a PS, may allow (i) an increase in the anticancer effects of the drug, and (ii) a decrease in its dose, and thus, its dose-related adverse effects.

Keywords: doxorubicin; photoactivation; photochemotherapy; photodynamic therapy; photosensitizer; reactive oxygen species.

Scheme 1

Chemical structure of doxorubicin.

Figure 1

Redox cycling of doxorubicin. One-electron reduction of the quinone moiety mediated by the cellular oxidoreductases results in the formation of a semiquinone radical that regenerates the parent quinone by reducing an oxygen molecule to a superoxide radical. This initiates a reaction cascade leading to the formation of other ROS.

Figure 2

(A) A 3D representation of the most stable tautomer of the doxorubicin molecule. (B) Jablonski diagram of doxorubicin. (C) HOMO and LUMO of doxorubicin. (D) Experimental (red line) and calculated UV–Vis spectra of doxorubicin.

Figure 3

Quantification of ROS generated through type I (A) and type II (B) mechanisms as a function of doxorubicin concentration after 30 min of white light irradiation. (A) Amplex Red assay for H₂O₂ detection. (B) ABMDMA assay for ¹O₂ detection. Results are expressed as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control (doxorubicin 0 μM).

Figure 4

Percentage (%) of Jurkat (A), A-431 (B), MCF-7 (C), and MDA-MB-231 (D) viable cells after treatment with doxorubicin for 2.5 h followed or not by 30 min light irradiation and 24 h recovery in drug-free complete medium. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. untreated cells. # p < 0.05; ## p < 0.01; ### p < 0.001 vs. nonirradiated cells.

Figure 5

Cellular uptake and nuclear colocalization of doxorubicin in MDA-MB-231 and Jurkat cells treated with doxorubicin (20 μM) for 0, 0.5, 1, 1.5, 2, and 2.5 h. (A,C) confocal microscopy images of internalized doxorubicin (magenta) and nuclei (cyan) at different time points. (B,D) scatter plot of doxorubicin nuclear colocalization calculated by the colocalization finder plug-in for ImageJ, r is Pearson’s correlation coefficient and r = 1 indicates complete colocalization.

Figure 6

Caspase-3 activity (A) and percentage (%) of SYTOX™ Green-positive cells (B) in Jurkat and MDA-MB-231 cells after treatment with doxorubicin (20 μM) for 2.5 h followed or not by light irradiation for 30 min and 24 h recovery in drug-free complete medium. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. untreated cells. # p < 0.05; ## p < 0.01 vs. nonirradiated cells.

Figure 7

ROS generation in Jurkat and MDA-MB-231 cells after treatment with doxorubicin 20 or 40 µM for 2.5 h followed or not by light irradiation for 30 min. Results are expressed as mean ± SD. *** p < 0.001; **** p < 0.0001 vs. untreated cells; ### p < 0.001; #### p < 0.0001 vs. the same concentration of doxorubicin in dark conditions.