COUPY Coumarins as Novel Mitochondria-Targeted Photodynamic Therapy Anticancer Agents

Abstract

Photodynamic therapy (PDT) for cancer treatment has drawn increased attention over the last decades. Herein, we introduce a novel family of low-molecular-weight coumarins as potential PDT anticancer tools. Through a systematic study with a library of 15 compounds, we have established a detailed structure-activity relationship rationale, which allowed the selection of three lead compounds exhibiting effective in vitro anticancer activities upon visible-light irradiation in both normoxia and hypoxia (phototherapeutic indexes up to 71) and minimal toxicity toward normal cells. Acting as excellent theranostic agents targeting mitochondria, the mechanism of action of the photosensitizers has been investigated in detail in HeLa cells. The generation of cytotoxic reactive oxygen species, which has been found to be a major contributor of the coumarins' phototoxicity, and the induction of apoptosis and/or autophagy have been identified as the cell death modes triggered after irradiation with low doses of visible light.

Figure 1

General structure of the classical coumarin scaffold and of coumarin-based COUPY derivatives.

Figure 2

Structure of COUPY derivatives 1–15 investigated.

Scheme 1. Synthesis of COUPY Derivatives 1–15

The structure of the compounds is shown in Figure 2.

Figure 3

Cellular uptake of coumarins 1, 2, 8, and 15. Single confocal planes of HeLa cells incubated with the compounds (0.5 μM for 1, 1 μM for 2 and 15, and 5 μM for 8,) for 30 min at 37 °C. Excitation was performed with the 561 nm laser and emission detected from 570 to 670 nm. White arrows point out mitochondria, white arrowheads nucleoli, and yellow arrowheads vesicle staining. Scale bar: 20 μm. LUT: Fire.

Figure 4

ROS levels in HeLa cells upon irradiation treatments with 5 μM of compounds 1, 2, and 15 (1 h in the dark followed by 1 h under light irradiation). Statistical significance from treated cells based on *p < 0.05, **p < 0.01, and ***p < 0.001 using the unpaired t-test. Data represented as mean ± SD (n = 3 replicates).

Figure 5

(a) Number of autophagic vesicles in HeLa cells after irradiation treatments as quantified by confocal microcopy imaging through monodansylcadaverine (MDC) staining. (S: Starvation for 2 h; R: resveratrol 50 μM). Data represented the mean ± SD from >10 cells from two independent experiments. (b–d) IC₅₀ values (mean ± SD) in HeLa cells for 1, 2, and 15, respectively, in the dark, after irradiation (0.5 h incubation, 1 h irradiation with visible light, and 48 h of recovery) or pretreated with wortmannin (50 μM) for 1 h prior irradiation schedule. Statistical significance from irradiation treatments based on *p < 0.05, **p < 0.01, and ***p < 0.001 using the unpaired t-test.

Figure 6

Flow cytometry evaluation of cell death induction in HeLa cells upon treatment with coumarins (5 μM for 1 and 2; 0.5 μM for 15) after irradiation treatments as revealed by dual Annexin V/propidium iodide labeling. Cisplatin (30 μM) used as a positive control for cell death induction. Data represented as mean ± SD and statistical significance from dark/irradiated conditions based on *p < 0.05, **p < 0.01, and ***p < 0.001 obtained using the unpaired t-test.