Photodynamic Therapy against Colorectal Cancer Using Porphin-Loaded Arene Ruthenium Cages

Abstract

Colorectal cancer (CRC) is the third most common cancer in the world, with an ongoing rising incidence. Despite secure advancements in CRC treatments, challenges such as side effects and therapy resistance remain to be addressed. Photodynamic therapy (PDT) emerges as a promising modality, clinically used in treating different diseases, including cancer. Among the main challenges with current photosensitizers (PS), hydrophobicity and low selective uptake by the tumor remain prominent. Thus, developing an optimal design for PS to improve their solubility and enhance their selective accumulation in cancer cells is crucial for enhancing the efficacy of PDT. Targeted photoactivation triggers the production of reactive oxygen species (ROS), which promote oxidative stress within cancer cells and ultimately lead to their death. Ruthenium (Ru)-based compounds, known for their selective toxicity towards cancer cells, hold potential as anticancer agents. In this study, we investigated the effect of two distinct arene-Ru assemblies, which lodge porphin PS in their inner cavity, and tested them as PDT agents on the HCT116 and HT-29 human CRC cell lines. The cellular internalization of the porphin-loaded assemblies was confirmed by fluorescence microscopy. Additionally, significant photocytotoxicity was observed in both cell lines after photoactivation of the porphin in the cage systems, inducing apoptosis through caspase activation and cell cycle progression disruptions. These findings suggest that arene-Ru assemblies lodging porphin PS are potent candidates for PDT of CRC.

Keywords: apoptosis; arene-ruthenium assemblies; colorectal cancer; photodynamic therapy; photosensitizers.

Figure 1

Schematic description of the cellular mechanisms induced by PS⸦M1 and PS⸦M2, evaluated in this study. (Created with BioRender.com (accessed on 29 June 2024)).

Figure 2

Chemical structure of porphin (PS) and the arene-ruthenium assemblies M1 and M2, together with the host–guest systems PS⸦M.

Figure 3

Phototoxicity of arene-Ru porphin PS assemblies on human CRC cell lines. (A) HCT116 and (B) HT-29 cell lines were cultured in RPMI medium for 24 h. After 24 h, cells were treated or not with PS⸦M1, PS⸦M2, M1, or M2. Illumination (630 nm, 75 J/cm²) of the cells occurred 24 h after treatment, and the cell viability for all the conditions was determined 12 h, 24 h, and 48 h post-illumination. Data are represented as a mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; and *** p < 0.001. (C) Graphical representation of the IC₅₀ values (nM) of PS⸦M1 and PS⸦M2 determined by MTT assay on HCT116 and HT-29 cell lines. Data are represented as a mean ± SEM of three independent experiments.

Figure 3

Phototoxicity of arene-Ru porphin PS assemblies on human CRC cell lines. (A) HCT116 and (B) HT-29 cell lines were cultured in RPMI medium for 24 h. After 24 h, cells were treated or not with PS⸦M1, PS⸦M2, M1, or M2. Illumination (630 nm, 75 J/cm²) of the cells occurred 24 h after treatment, and the cell viability for all the conditions was determined 12 h, 24 h, and 48 h post-illumination. Data are represented as a mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; and *** p < 0.001. (C) Graphical representation of the IC₅₀ values (nM) of PS⸦M1 and PS⸦M2 determined by MTT assay on HCT116 and HT-29 cell lines. Data are represented as a mean ± SEM of three independent experiments.

Figure 4

Intracellular ROS production was evaluated in (A) HCT116 and (B) HT-29 human CRC cell lines immediately after illumination (630 nm, 75 J/cm²) using DCFDA staining. Cells were analyzed using flow cytometry. Quantification of the intensity of fluorescence emitted due to DCF formation is correlated to the level of ROS generation. Data are represented as a mean ± SEM of three independent experiments. ** p < 0.01 and *** p < 0.001.

Figure 5

Detection of the cellular internalization of PS⸦M1 and PS⸦M2 in HCT116 (A) and HT-29 (B) cell lines by confocal microscopy. The cells were seeded into incubation chambers and cultured for 24 h. The cells were then treated with the compounds, and the fluorescence was measured by confocal microscopy (laser Zeiss LSM 510 Meta―×1000). The internalization was processed using the ImageJ image-processing software (version 1.54f). White scale bar = 20 μm.

Figure 6

Cell cycle distribution analysis on HCT116 and HT-29 cell lines after the photoactivation of PS⸦M1 and PS⸦M2. Cells were seeded for 24 h in a culture medium before the treatment or not with the assemblies at their determined IC₅₀ concentrations. After 24 h of treatment, cells were illuminated or not with red light at 630 nm and 75 J/cm². Cells were collected at 12 h, 24 h, and 48 h post-illumination for analysis by flow cytometry using PI staining. Images of the cell cycle distribution on (A) HCT116 cells at 24 h post-illumination and (B) HT-29 cell line at 48 h post-illumination are represented. Histograms representing the percentage cell numbers at each phase of the cell cycle on (C) HCT116 and (D) HT-29 cell lines are displayed as a mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01 and *** p < 0.001.

Figure 7

Apoptosis due to photoactivation was assessed on HCT116 and HT-29 cell lines. Cells were seeded and incubated for 24 h then treated or not with PS⸦M1 and PS⸦M2 assemblies at IC₅₀ concentrations. Cells were either illuminated (630 nm, 75 J/cm²) or not after 24 h of treatment, and then, they were collected at 12 h, 24 h, and 48 h post-illumination. The collected cells were stained with Annexin V- FITC and PI, and their state was revealed by flow cytometry. Representative data from flow cytometry for the HCT116 cell line at 24 h post-illumination (A) and HT-29 at 48 h post-illumination (B) are displayed. Histograms represent the viable and apoptotic cell percentages of the treated HCT116 (C), and HT-29 (D) cells subjected to prior illumination. Data are represented as a mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Figure 7

Apoptosis due to photoactivation was assessed on HCT116 and HT-29 cell lines. Cells were seeded and incubated for 24 h then treated or not with PS⸦M1 and PS⸦M2 assemblies at IC₅₀ concentrations. Cells were either illuminated (630 nm, 75 J/cm²) or not after 24 h of treatment, and then, they were collected at 12 h, 24 h, and 48 h post-illumination. The collected cells were stained with Annexin V- FITC and PI, and their state was revealed by flow cytometry. Representative data from flow cytometry for the HCT116 cell line at 24 h post-illumination (A) and HT-29 at 48 h post-illumination (B) are displayed. Histograms represent the viable and apoptotic cell percentages of the treated HCT116 (C), and HT-29 (D) cells subjected to prior illumination. Data are represented as a mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Figure 8

Protein expression was evaluated by Western blot. HCT116 (A) and HT-29 (B) cells were seeded and incubated for 24 h and then were treated or not at IC₅₀ values of PS⸦M1 or PS⸦M2. After treatment, cells were either illuminated (630 nm, 75 J/cm²) or not and collected 24 h and 48 h post-illumination. Proteins were extracted, and the level of protein expression of the different conditions were revealed. β-actin was used as a loading control. Representative images are shown.

Figure 9

DNA fragmentation in HCT116 (A) and HT-29 (B) cells analyzed from cytosol extracts using ELISA assay. After seeding the cells for 24 h, followed by treatment, the cells were illuminated (630 nm, 75 J/cm²) or not. After 12 h, 24 h, and 48 h post-illumination, the cells were collected, and the level of DNA fragmentation was analyzed. Histograms are represented as a mean ± SEM of at least three independent experiments. ** p < 0.01 and *** p < 0.001.