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

Abstract

Background and Objectives 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. Methods 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. Results 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%. Conclusions 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. halepensis bark extract showed the potential to be used as a supplementary agent to improve prostate cancer PDT treatment.

The Photosensitizer Temoporfin (mTHPC) – Chemical, Pre‐clinical and Clinical Developments in the Last Decade†‡

“…The combination allowed to eliminate the tumor in this murine prostate cancer model while at the same time reducing photosensitivity. Similar protective effects of P. halepensis extracts were observed in in vitro experiments in the LNCaP prostate cancer cell line (176). Korbelik et al .…”

“…PDT is associated with side effects like skin photosensitization. In this respect the photoprotective properties of plant extracts have been investigated (176,291,579). In investigations with a plant extract from P. halepensis the authors reported that the extract prevented photosensitivity after temoporfin PDT in mice (291) and even increased intracellular ROS levels (176).…”

The effects of low power laser light at 661 nm on wound healing in a scratch assay fibroblast model

“…ROS levels were quantified according to the method described in [ 19 ]. In brief, images were converted to 8 bit grayscale ones.…”

Responsive Peptide Nanofibers with Theranostic and Prognostic Capacity

“…In addition to locating the tissue of interest, it is often possible to discern areas of pre‐existing necrosis, and areas of disease infiltration by choosing from a variety of techniques or contrast agents [26, 27] . The structural and functional information gathered through pretreatment imaging can be used in treatment planning, such as the location of the light source, and even treatment dose assessment [28] . Moreover, imaging techniques have been successfully clinically implemented that allow for the assessment of the efficacy of PDT by comparing tissue images before and after treatment, showing evidence of necrosis, apoptosis, and blood vessel occlusion [28, 29] .…”

“…[26,27] The structural and functional information gathered through pretreatment imaging can be used in treatment planning, such as the location of the light source, and even treatment dose assessment. [28] Moreover, imaging techniques have been successfully clinically implemented that allow for the assessment of the efficacy of PDT by comparing tissue images before and after treatment, showing evidence of necrosis, apoptosis, and blood vessel occlusion. [28,29] The development of photosensitizer-based nanostructures with intrinsically built-in fluorescence imaging capacity, would integrate these features in one particle, which would allow for facile monitoring of accumulation and retention at the tumor site before and after PDT, and assess the progress of PDT through in situ real-time imaging without using additional contrast agent.…”