Epigallocatechin Gallate Enhances MAL-PDT Cytotoxic Effect on PDT-Resistant Skin Cancer Squamous Cells

Abstract

Photodynamic therapy (PDT) has been used to treat certain types of non-melanoma skin cancer with promising results. However, some skin lesions have not fully responded to this treatment, suggesting a potential PDT-resistant phenotype. Therefore, novel therapeutic alternatives must be identified that improve PDT in resistant skin cancer. In this study, we analyzed the cell viability, intracellular protoporphyrin IX (PpIX) content and subcellular localization, proliferation profile, cell death, reactive oxygen species (ROS) detection and relative gene expression in PDT-resistant HSC-1 cells. PDT-resistant HSC-1 cells show a low quantity of protoporphyrin IX and low levels of ROS, and thus a low rate of death cell. Furthermore, the resistant phenotype showed a downregulation of HSPB1, SLC15A2, FECH, SOD2 and an upregulation of HMBS and BIRC5 genes. On the other hand, epigallocatechin gallate catechin enhanced the MAL-PDT effect, increasing levels of protoporphyrin IX and ROS, and killing 100% of resistant cells. The resistant MAL-PDT model of skin cancer squamous cells (HSC-1) is a reliable and useful tool to understand PDT cytotoxicity and cellular response. These resistant cells were successfully sensitized with epigallocatechin gallate catechin. The in vitro epigallocatechin gallate catechin effect as an enhancer of MAL-PDT in resistant cells is promising in the treatment of difficult skin cancer lesions.

Keywords: methyl aminolevulinate; non-melanoma skin cancer; photodynamic therapy; squamous cell carcinoma.

Figure 1

Generation of photodynamic therapy (PDT) resistance in HSC-1 cells. (A) The figure represents the workflow used to obtain a resistant population after 10 cycles of PDT. (B) Cell viability of parental HSC-1 cells compared to PDT-resistant HSC-1 cells at different light doses. (C) Lethal dose and fold-change index calculated for each cell line. Values of p < 0.05 were considered statistically significant. LD = Lethal dose; * p < 0.05. Data were expressed as mean ± SD of three biological replicates.

Figure 2

Cell death and proliferation capacity analysis. (A) Cell death analysis (AV (+) and PI (+)); (B) PS translocation (AV (+)). (C) Representative plot of the flow cytometry for cell death and PS translocation assays in parent and resistant cells. (D) Clonogenic assay, 500 cells were seeded in each plate for 14 days. (E) Quantification of colonies formed in parental HSC-1 and resistant cells. (F) Representative images of wound closure in parental HSC-1 and resistant cells. (G) Percentage of wound closure in resistant cells compared to parental HSC-1. Values of p < 0.05 were considered statistically significant. * p < 0.05; *** p < 0.001. Data were expressed as mean ± SD of three biological replicates.

Figure 3

Production of PpIX and ROS in PDT-resistant HSC-1 cells. (A) PpIX was found mainly in the cytoplasm of both parental HSC-1 and resistant cells. Nuclei are stained with DAPI (Blue), while PpIX fluorescing in red under blue exciting light (λEx = 460–490 nm). (B) Positive cells for PpIX production. (C) PpIX fluorescence intensity in parental HSC-1 compared to resistant cells at different times. (D) Intracellular content of PpIX measured at λ_Ex 406 and λ_Em 605 nm using a spectrophotometer. (E) Fluorescence intensity of ROS production in parental HSC-1 and resistant cells. Values of p < 0.05 were considered statistically significant. a.u. = arbitrary units; * p < 0.05; ** p < 0.01; *** p < 0.001. Data were expressed as mean ± SD of three biological replicates.

Figure 4

(A) Transcriptional expression of genes associated with membrane transporters, cell metabolism (enzymes), cell stress, hypoxia and cell survival in HSC-1-parental and resistant cells. (B) Protein expression of HSP27 and survivin in parental and PDT-R HSC-1. Values of p < 0.05 were considered statistically significant. * p < 0.05. Data were expressed as mean ± SD of three biological replicates.

Figure 5

Combined effect of PDT and EGCGT in PDT-resistant HSC-1 cells. (A) Cell viability in PDT-resistant HSC-1 cells treated with EGCG. (B) Positive cells for PpIX production in PDT-resistant HSC-1 cells treated with MAL and EGCG at different concentrations. (C) PpIX fluorescence intensity in PDT-resistant HSC-1 cells treated with MAL and EGCG at different concentrations. (D) ROS production in PDT-resistant HSC-1 cells with combined therapy (PDT+EGCG). Each experimental group was compared with their respective control, either MAL (2 mM) or MAL (2 mM) + Light (4 J/cm²). Values of P < 0.05 were considered statistically significant. a.u. = arbitrary units; ** p < 0.01. Data were expressed as mean ± SD of three biological replicates.