Assessment of natural antioxidants' effect on PDT cytotoxicity through fluorescence microscopy image analysis.

Abstract

Photodynamic therapy (PDT) is a cancer treatment modality mediated by reactive oxygen species (ROS). However, the intracellular antioxidant defense system antagonizes PDT-generated ROS, impeding PDT efficacy. This study aimed to evaluate the enhancement of PDT cytotoxicity by its combination with natural antioxidants in pro-oxidant concentrations. A rich natural antioxidant mixture originating from Pinus halepensis bark extract was studied for its potential to enhance the efficacy of m-tetrahydroxyphenylchlorin (m-THPC)-PDT on LNCaP prostate cancer cells, in vitro. Various P. halepensis concentrations, at two different incubation times, were used in combination with m-THPC-PDT. Assessment of cellular viability and intracellular ROS levels evaluated the treatments' outcome. A novel method was developed for the assessment of the intracellular ROS levels, based on image analysis and data extraction from fluorescence microscopy images. P. halepensis bark extract increased the intracellular ROS levels in a concentration-dependent but not in an incubation-dependent manner. The higher concentrations used (≥50 μg/ml) reduced cellular viability even by 50%. One hour pretreatment with 30 μg/ml P. halepensis before m-THPC-PDT exceeded the levels of cellular death by approximately 15%. The results provided evidence of the cytotoxic effect of P. halepensis bark extract on LNCaP cells, showing the potential of P. halepensis to be used as an anticancer agent in prostate cancer treatment. The results also provided evidence of enhancement of m-THPC-PDT by P. halepensisbark extract showed the potential to be used as a supplementary agent to improve prostate cancer PDT treatment.