Photodynamic therapy (PDT), locally applied to solid C6 rat glioma tumors in the foot of CD1 nude mice, eradicated the primary tumor and also decreased the rate of groin and lung metastases. Pd-Bacteriopheophorbide (Pd-Bpheid), a novel photosensitizer synthesized in our laboratory, was used in our study. The primary lesion in the hind leg was treated by an i.v. injection of 5 mg/kg of Pd-Bpheid and immediate illumination (650 -800 nm, 360 J/cm 2 ). This protocol and the surgical amputation of the leg were compared for local and metastasis responses. Following PDT, hemorrhage, inflammation with tumor necrosis and flattening were observed and histologically verified in the photodynamically treated tumor. Whereas local tumor control rates were up to 64% following PDT, in surgically treated animals, local tumor control was absolute. The rates of metastases in the groin and the lungs were at least 12-fold lower in the photodynamically treated animals compared with untreated or surgerytreated groups. The overall cure rates after PDT or surgery were 36% and 6%, respectively, at 8 weeks. These findings suggest that local PDT with Pd-Bpheid, which acts primarily on the tumor vasculature, efficiently eradicates the solid C6 tumors. In addition, the local PDT of the primary lesion has beneficial therapeutic effects on remote C6 metastasis, which is not obtained with surgery. It is therefore suggested, that although surgery is highly efficient for the immediate removal of the primary tumor, it lacks such systemic, therapeutic effects on distant metastases. Pd-Bpheid-PDT may thus offer a potentially superior curative therapy for C6 glioma tumors in the limb by eradicating the target tumor and by reducing the rate of metastasis in the groin and lung, possibly due to innate immunity. © 2002 Wiley-Liss, Inc. Key words: photodynamic therapy; surgery; Pd-bacteriopheophorbide; C6 glioma tumor; metastasisPhotodynamic therapy (PDT) is based on the destruction of tumors by cytotoxic reactive oxygen species (ROS) produced upon local tumor illumination in patients administered with a photosensitizer. 1-3 Following health agency approval for photofrin-based PDT in many countries, this anti-cancer treatment modality entered clinical use for the local treatment of an increasing number of indications including skin, esophageal, lung, gastric, cervical and bladder cancers. 4 PDT is usually considered a local anti-tumor treatment modality. However, reports from several laboratories suggest that PDT also induces beneficial systemic effects. Following in vitro hematoporphyrin-based PDT, adhesiveness and metastatic potential decline in DHD-K12-cultured colon carcinoma cells. Moreover, intravenous or s.c. injection of these PDT-treated cells to rats resulted in a reduced number of lung metastases compared with untreated cell injection. 5,6 Although this observation may be due to local photodynamic damage, the potential beneficial effect may be viewed as systemic. Other in vivo studies showed that local PDT with various photosensitizers mediates ...

Mn-superoxide dismutase (Mn-SOD), which protects the cell from the toxic potential of superoxide radicals (O(2)(-*)), is the only type of SOD which resides in eukaryotic mitochondria. Up-to-date, the exact catalytic mechanism of the enzyme and the relationship between substrate moieties and the ligands within the active site microenvironment are still not resolved. Here, we set out to explore the possible involvement of hydroperoxyl radicals ((*)OOH) in the catalytic dismutaion by following the interplay of Mn(III)/Mn(II) redox transitions, ligands binding, and evolution or consumption of superoxide radical, using a new model system. The model system encompassed an Mn atom chelated by a bacteriochlorophyll allomer macrocycle (BChl) in aerated aprotic media that contain residual water. The redox states of the Mn ion were monitored by the Q(y) electronic transitions at 774 and 825 nm for [Mn(II)]- and [Mn(III)]-BChl, respectively (Geskes, C.; Hartwich, G.; Scheer, H.; Mantele, W.; Heinze, J. J. Am. Chem. Soc. 1995, 117, 7776) and confirmed by electron spin resonance spectroscopy. Evolution of (*)OOH radicals was monitored by the ESR spin-trap technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The experimental data suggest that the [Mn]-BChl forms a (HO(-))[Mn(III)]-BChl(OOH) complex upon solvation. Spectrophotometeric titrations with tetrabutylamonnium acetate (TBAA) and 1-methylimidazole (1-MeIm) together with ESI-MS measurements indicated the formation of a 1:1 complex with [Mn]-BChl for both ligands. The coordination of ligands at low concentrations to [Mn(III)]-BChl induced a release of a (*)OOH radical and a [Mn(III)]-BChl --> [Mn(II)]-BChl transition at higher concentrations. The estimated equilibrium constants for the total redox reaction ( )()are 1.9 x 10(4) +/- 1 x 10(3) M(-)(1) and 12.3 +/- 0.6 M(-)(1) for TBAA and 1-MeIm, respectively. The profound difference between the equilibrium constants agrees with the suggested key role of the ligand's basicity in the process. A direct interaction of superoxide radicals with [Mn(III)]-BChl in a KO(2) acetonitrile (AN) solution also resulted in [Mn(III)]-BChl --> [Mn(II)]-BChl transition. Cumulatively, our data show that the Mn(III) center encourages the protonation of the O(2)(-)(*) radical in an aprotic environment containing residual water molecules, while promoting its oxidation in the presence of basic ligands. Similar coordination and stabilization of the (*)OOH radical by the Mn center may be key steps in the enzymatic dismutation of superoxide radicals by Mn-SOD.

Various forms of cellular stress induce adaptive responses through poorly understood mechanisms. In maintaining homeostasis, endothelial cells respond and adapt to changes in oxidative stress that prevail in the circulation. Endothelial cells are also the target of many oxidative stress-based vascular therapies. The objectives of this study were to determine whether endothelial cells adapt to oxidative stress induced upon the photosensitization of WST11 (a water-soluble Pd-bacteriochlorophyll derivative being developed as a photodynamic agent) and to study possible cellular mechanisms involved. The hallmark of WST11-based photodynamic therapy is the in situ generation of cytotoxic reactive oxygen species causing vascular shutdown, hypoxia, and tumor eradication. Here we demonstrated that photodynamic therapy also induces adaptive responses and tolerance following a sublethal preconditioning of endothelial cells with the same (homologous) or different (heterologous) stressor. A link among p38 MAPK activity, expression of hsp70 and hsp27, and homologous adaptation to reactive oxygen species induced by photosensitized WST11 was established. In addition to characterization of some key proteins involved, our observations provide a beneficial new working tool for the studies of mechanisms involved in oxidative stress and adaptation using light-controlled photosensitization.Oxidative stress can trigger two opposing cellular responses depending on the severity of the induced stress, one leading to cell death and the other to transient non-lethal physiological changes. A major feature of the physiological response to oxidative stress is its adaptive and protective nature. Adaptation or tolerance to stress can be defined as the ability of a cell or an organism to become resistant to stress following a sublethal stress experience (1). For instance, clinically relevant adaptation has been mentioned with respect to protection of the heart myocardium and other organs against ischemia and reperfusion injury (2). The adaptation process is time-dependent and requires physiological rearrangement. Evidently, if cells are sensitized by oxidative stress at low levels, tolerance to a second oxidative challenge will probably be manifested within 16 -24 h (3).Oxidative stress is the basis of photodynamic therapy (PDT) 1 where tumors are destroyed by an overwhelming burst of cytotoxic reactive oxygen species (ROS) generated upon local in situ photosensitization of an administered photosensitizer (4). In situ generation of ROS by photosensitization of preaccumulated pigments in cultured tumor cells has been used for the elucidation of the molecular basis of PDT (5). Endothelial cells (ECs) serve as a major target in anti-vascular PDT induced by bacteriochlorophyll derivatives (6 -12). Furthermore, ECs are most sensitive to rapid oxidative changes in the circulation and are presumably capable of adapting to these changes. Consequently, cultured H5V mouse ECs were chosen as a model in this study of adaptation to oxidative stress.The basis ...