Photodynamic therapy with hypericin induces vascular damage and apoptosis in the RIF‐1 mouse tumor model

Abstract

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been proven to be a potent photosensitizer. The mechanism of tumor eradication and mode of cell death induced by in vivo photodynamic therapy (PDT) with hypericin were investigated in the present study using 2 therapeutic protocols. RIF-1 tumors were exposed to laser light at either 0.5 hr or 6 hr after hypericin administration (5 mg/kg, i.v.). A significant reduction in tumor perfusion, as determined by the retention of fluorescein in the tumor tissue, was detected immediately after both PDT treatments. Further decrease in tumor perfusion was observed in the hours after treatment. The re-establishment of tumor perfusion, however, occurred 24 hr after 6 hr-interval PDT, but not after 0.5 hr-interval PDT. The kinetics of tumor cell survival estimated by the in vivo/in vitro clonogenic assay revealed no or limited cell death when tumors were explanted immediately after irradiation, whereas a delayed but progressive cell death was detected when tumors remained in situ after both PDT treatments. The detection of nucleosomal DNA fragmentation by agarose gel electrophoresis or TUNEL assay and the assessment of cell morphology by light microscopy indicated that apoptosis was the most prominent tumor response to hypericin-mediated PDT. Furthermore, immunohistochemical analysis of the tumor tissue showed an increased expression of both Fas and Fas ligand after irradiation, suggesting that this cell death pathway might contribute to the overall PDT-induced apoptotic response. In conclusion, our results demonstrate that apoptosis, likely occurring as a result of vascular damage, is responsible for the tumor eradication by PDT with hypericin in this tumor model.

Elucidation of the tumoritropic principle of hypericin

“…We have recently been focusing on hypericin, a natural compound isolated from Hypericum plants (Lavie et al, 1995a), as a potent photosensitiser with a high antitumoral PDT efficacy (Chen and de Witte, 2000;Chen et al, 2001Chen et al, , 2002. In the course of our study, we found that the compound accumulated to a large extent in tumour tissues.…”

“…The RIF-1 and R1 tumour-bearing animals were used to quantify the tumour perfusion by means of spectrofluorometric determination of tumour FITC-dextran uptake (fluorescein isothiocyanate dextran, M r 2 Â 10 6 , obtained from Sigma, St Louis, MO, USA), as described (Chen et al, 2002).…”

Photodynamic therapy and anti-tumour immunity

“…There is an extensive body of literature that examines the pathways of apoptosis that are induced after PDT in both normal and tumour cells in tissue culture, such as signalling pathways 30,31 , mitochondrial events 4 and mediators of apoptosis 32 . The occurrence of apoptosis after PDT in tumours in vivo has also been shown [33][34][35] , but there have been no studies looking at in vivo clearance mechanisms of apoptotic cells in tumours after PDT.…”

“…There is an extensive body of literature that examines the pathways of apoptosis that are induced after PDT in both normal and tumour cells in tissue culture, such as signalling pathways 30,31 , mitochondrial events 4 and mediators of apoptosis 32 . The occurrence of apoptosis after PDT in tumours in vivo has also been shown [33][34][35] , but there have been no studies looking at in vivo clearance mechanisms of apoptotic cells in tumours after PDT.Many studies have examined the relationship between the mode of tumour cell death (by methods other than PDT) and the efficiency of induction of the immune response, both in 36,37 . Although some reports show that apoptotic tumour cells are more effective than necrotic tumour cells at inducing an immune response 38,39 , there are other reports that show that modes of cancer therapy that predominantly induce necrosis are actually better at activating the immune system than methods that predominantly induce apoptosis 40,41 .…”

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

“…72 Antibodies against LC3 (3868) and SERCA2 (4388) were from Cell Signaling Technology. Anti-Cyt C antibody (556433) Figure 4D and E. Secondary ROS modulate organelle-specific autophagy.…”