Grada Kulikova, Lucia (2009) Different effects of single and fractionated light delivery regimes in photodynamic therapy with hypericin on HT-29 cells in vitro. [Tesi di dottorato] (Unpublished)

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Photodynamic therapy (PDT) is an alternative cancer cure which involves the selective uptake and retention of a photosensitizer in a cancer tissue, followed by irradiation with light of a specific wavelength to kill tumour cells via the production of reactive oxygen species (ROS). Fractionation of the light administration is one of the protocol modifications in PDT based on the hypothesis of tissue reoxygenation during the one or more dark intervals between illuminations with a subsequent greater production of singlet oxygen and thus greater PDT effect. However we demonstrate in the present study that two-fold illumination scheme with equal light doses (3 or 6 J.cm-2) separated by a dark interval 1 or 6 h do not enhanced, even reduced hypericin-mediated photocytotoxic effect in HT-29 adenocarcinoma cells in vitro by illumination with unequal light doses (1 + 11 J.cm-2) separated with a longer dark pause (6 h). Fractionation with a longer dark pause increased cell number and cell survival in HT-29 cells when compared with such treatment but with a 1 h dark pause or with a single light delivery (12 J.cm-2). Even proportion of cells in G1 and G2 phase of cell cycle were near to control. Longer dark pause also repressed cell death and enhanced clonogenic potential of HT-29 cells. Since longer dark interval after the irradiation by first sub-lethal light dose (1 J.cm-2) makes cells resistant to the effect of the lethal light dose (11 J.cm-2), we studied the events proceeded during a dark pause after sub-lethal dose (1 J.cm-2) up to the second illumination. We show that pre-sensitization did not affect physiological elimination of hypericin however administration regime affected hypericin elimination after lethal dose. Inhibition of p38 MAPK did not improve photocytotoxic effect of light fractionation. Cell pre-sensitization induced ROS production, increased activity of redox-regulated nuclear transcription factor NF-κB and expression of proteins connected with a cell survival (NF-κB p50 and p65 subunits, IκB-α, Mcl-1, HSP70, GRP94, Clusterin-α). Although the role of heat shock proteins was generally established in response upon photo-induced stress, we uncovered that HSPs are not necessarily the “key” molecules in photo-resistance. Our findings indicate that timing of the second light dose before or after NF-κB activation could be crucial for the fate of cancer cell. We estimate successful application of hypericin in a high-dose multi-fraction PDT with dark intervals reduced bellow 1 h that might yield improved outcome.

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