Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy.

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.

Pre-treatment of HT-29 cells with 5-LOX inhibitor (MK-886) induces changes in cell cycle and increases apoptosis after photodynamic therapy with hypericin.

It may be hypothesized that the lipoxygenase (LOX) metabolic pathway plays an important role in photodynamic therapy (PDT) of malignant tumours, and modification of this pathway may result in administration of lower doses of photodynamic active agents accompanied by reduced side effects. In this study, we examine in more detail the cytokinetic parameters of human colon adenocarcinoma HT-29 cells pre-treated for 48 or 24h with LOX inhibitor MK-886, followed by PDT induced by hypericin. Based on MTT assay the concentrations of both agents (MK-886 and hypericin) with relatively slight (non-significant) cytotoxic effects were selected. These concentrations were used for combined treatment, where MTT response, total cell number, floating cells quantification, viability, cell cycle progression and DNA synthesis were detected. Hoechst/PI staining, PARP fragmentation and mitochondrial membrane potential (MMP) were evaluated to determine the extent of apoptosis. While MK-886 alone caused mainly necrosis, 48h pre-treatment of cells with MK-886 followed by PDT with hypericin clearly shifted the type of cell death to apoptosis. PDT with hypericin alone caused apoptosis in 19% of the cell population. Some combined modalities significantly potentiated the apoptotic effect (31% of apoptotic cells; 2.5microM MK-886/0.1microM hypericin), i.e., by 60% more than after single treatment with hypericin. Increased apoptosis was confirmed by PARP (116kDa) cleavage to characteristic 89kDa fragments and changes in MMP. Increasing concentration of MK-886 was accompanied by massive changes in the cell cycle progression. Combined treatment with lower concentrations of MK-886 and hypericin increased accumulation of cells in the S phase, accompanied by inhibition of DNA synthesis. Increasing concentration of MK-886 in this combination caused the opposite effect, manifesting significant accumulation of cells in the G0/G1 phase. More pronounced effects were observed after the 48h pre-treatment schedule. This anti-proliferative effect was confirmed by BrdU incorporation. These results indicate that combined treatment involving PDT and LOX inhibitor MK-886 may improve the therapeutic effectiveness of PDT.

Effect of acetazolamide on hypericin photocytotoxicity.

The photodynamic action of hypericin (HYP) in vitro was evaluated using human leukemic HL-60 and lung carcinoma A549 cell lines. After illumination HYP (1 x 10 (-5) M) reduced the proliferation and/or survival of HL-60 and A549 cells vs. controls to almost to 0 % and 29 %, respectively. A lower concentration of HYP (1 x 10 (-6) M) decreased the proliferation and/or survival only in HL-60 cells. Non-cytotoxic concentrations of the carbonic anhydrase inhibitor acetazolamide (ACTZ) (1 x 10 (-3)-1 x 10 (-6) M) significantly potentiated these effects of HYP (1 x 10 (-6)M) in HL-60, but not in the A549 cell line. The highest concentration of ACTZ (1 x 10 (-3) M) also induced an increase of the subdiploid G 0 /G 1 population in HYP (1 x 10 (-6) M) treated HL-60 cells from 14 % to 24 %. The results indicate that the photogenerated pH drop may participate in the potentiation of the photodynamic action of HYP observed in leukemia cells.