Parental HSC-1 and resistant cells were lysed using a RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing Cilostazol protease and phosphatase inhibitor cocktail (1:100, Thermoscientific, Rockford, IL, USA). 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. < 0.05 were considered statistically significant. LD = Lethal dose; * < 0.05. Data were expressed as mean SD of Cilostazol three biological replicates. 2.2. Characterization of MAL-PDT-Resistant Phenotype in HSC-1 Cells In order to characterize the resistant cells according to cell death and proliferation process, we performed phosphatidylserine (PS) translocation (early event of apoptosis), cell death (late event of apoptosis), as well as clonogenic and wound healing assays. After Cilostazol one-hour post-treatment with MAL-PDT (4 J/cm2), parental HSC-1 cells showed a significant increase in PS translocation (8.63 7.13%) and cell death (36.96 7.08%) compared to PDT-resistant HSC-1 cells (1.06 0.87% and 9.04 2.21%, respectively) (Figure 2A,B, < 0.0005). Representative cytometric profiles according to cell death (Q1, Q2, and Q4) and PS translocation (Q4) are shown in Figure 2C. Open in a separate window 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 Cilostazol each plate for 14 days. (E) Quantification of colonies formed in parental HSC-1 and resistant cells. (F) Cilostazol 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 < 0.05 were considered statistically significant. * < 0.05; *** < 0.001. Data were expressed as mean SD of three biological replicates. According to cell proliferation capacity, we evaluated the ability to form colonies and wound healing. First, the colonies formed in both parental and PDT-resistant HSC-1 cells. PDT-resistant HSC-1 cells formed a higher number of colonies and larger than parental HSC-1 cells (Figure 2D,E). The wound healing assay showed that parental HSC-1 cells had 35.57 11.78% wound closure, while PDT-resistant HSC-1 cells closed the entire area at 36 h post-scratch (Figure 2F,G). These results show that resistant HSC-1 cells are capable of avoiding cell death and proliferating at a higher rate than parent cells. 2.3. PDT-Resistant HSC-1 Cells Show Lower Levels of PpIX and ROS than Parental Cells To determine the cellular location and intracellular content of PpIX, parental and resistant HSC-1 cells were observed by fluorescence microscopy after incubation with 2 mM of MAL. PpIX was located in the cytoplasm of both parental and resistant HSC-1 cells (Figure 3A). However, parental cells showed 80% positive cells for PpIX at 4 h post-incubation with MAL, while resistant cells showed about 20% (Figure 3B). Also, fluorescence intensity was higher in parental HSC-1 cells than resistant cells (Figure 3C). The intracellular content of PpIX was significantly higher in parental cells (81.81 4.41 ng/mg) than resistant cells (14.24 3.60 ng/mg) (Figure 3D). These findings suggest that parental HSC-1 cells synthesize and accumulate more PpIX than their resistant counterparts. Open in a separate window 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. Cxcl12 Nuclei are stained with DAPI (Blue), while PpIX fluorescing in red under blue exciting light (Ex = 460C490 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 Ex 406 and Em 605 nm using a spectrophotometer. (E) Fluorescence intensity of ROS production in parental HSC-1 and resistant cells. Values of < 0.05 were considered statistically significant. a.u. = arbitrary units; * < 0.05; ** < 0.01; *** < 0.001. Data were.