Rapid induction of apoptosis in human keratinocytes with the photosensitizer QLT0074 via a direct mitochondrial action.

QLT0074 is a newly introduced, porphyrin-derivative for use in photodynamic therapy (PDT). In the current study, the intracellular distribution of QLT0074 and the mode of cell death induced by photosensitization with this compound in vitro were assessed for transformed human HaCaT keratinocytes. Fluorescence microscopy studies indicated a distribution of the drug to the cytoplasm, nuclear membrane and mitochondria of these cells. In the absence of light, QLT0074 produced no evidence of apoptosis-related biochemical changes or affected cell viability. When combined with blue light exposure, cytotoxicity was exerted in a QLT0074- and light-dose-related manner. Appearance of the mitochondrial protein cytochrome c in the cytosolic fraction and expression of the apoptosis-associated mitochondrial 7A6 antigen were demonstrable following photosensitization at nano-molar levels of QLT0074. Evidence of processing of the apoptosis-effector molecules caspase-3, -6, -7, -8 and -9 as well as cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) were demonstrable subsequent to cytochrome c release after PDT. Treatment with the anti-oxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytochrome c release, caspase-3 activation and PARP cleavage associated with PDT thereby supporting the contention that QLT0074 induces apoptosis through the generation of reactive oxygen species upon light activation. QLT0074 is a potent photosensitizer with the capacity to directly initiate apoptosis by acting upon mitochondria.

Mitochondrial release of apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis.

Photodynamic therapy (PDT) is under investigation for the treatment of intimal hyperplastia in conditions such as atherosclerosis and restenosis. Although smooth muscle cells (SMCs) may be a key target for treatment, the effects of PDT on these cells are poorly characterized. In the present study, apoptosis was induced in primary human aortic SMCs by the combination of the photosensitizer verteporfin and visible light. After PDT, an increase in mitochondrial cytochrome c (cyt c) and apoptosis-inducing factor (AIF) levels were detected in the cytosol immediately and their levels increased steadily up to 2 hours. Cytosolic levels of the pro-apoptotic Bcl-2 family member Bax decreased reciprocally throughout this period, but this change did not occur before cyt c release. Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells as compared to a distinct mitochondrial staining in normal cells. AIF translocated from mitochondria to the nucleus during the progression of apoptosis. After cyt c release, caspase-9 and caspase-3 processing was visible by 1 hour and caspase-6, -7, and -8 processing was apparent by 2 hours after PDT. In summary, these results demonstrate for the first time the cellular redistribution of mitochondrial AIF during SMC apoptosis, as well as the early release of cyt c and the subsequent activation of multiple caspases during PDT-induced SMC apoptosis.

Benzoporphyrin derivative monacid ring A (Verteporfin) alone has no inhibitory effect on intimal hyperplasia: in vitro and in vivo results.

Benzoporphyrin derivative monoacid ring A (Verteporfin, BPD-MA), a photosensitizing drug, has been suggested as having inhibitory effects on smooth muscle cell (SMC) proliferation in rabbit aortic intimal injuries. The effect of BPD-MA on vascular SMCs in the absence of light stimulation in vitro and in vivo was studied using models of intimal hyperplasia. Human SMCs were incubated with BPD-MA for 4 h in darkness. A small (20%) but significant decrease in viability (n =42,p < .05) was noted for BPD-MA concentrations above 15 microg/mL. This was an all-or-none phenomenon with no further decrease in viability at higher concentrations. Treatment with BPD-MA was also carried out in vivo using a balloon injury model of intimal hyperplasia in rabbit aortas. Thirty-three rabbits were randomized into five groups and given intravenous BPD-MA (2 mg/kg) according to the following schedule: Group 1 (n = 8), BPD-MA 25 min prior to injury; Group 2 (n = 8), BPD-MA 25 min prior to injury plus a second dose 4 weeks later; Group 3 (n = 4), BPD-MA immediately postinjury; Group 4 (n = 7), BPD-MA immediately postinjury plus a second dose 4 weeks later; or Group 5 (n = 6), no drug (control group). No statistically significant difference was seen in the amount of intimal hyperplasia that developed in the five groups.

Reduced xenograft rejection in rat striatum after pretransplant photodynamic therapy of murine neural xenografts.

OBJECT: The goal of this study was to develop a method of reducing neural xenograft rejection by pretreating the graft with photodynamic therapy (PDT). METHODS: Xenograft cell suspensions were prepared from fetal mouse mesencephalon, after which they were incubated for 30 minutes with various concentrations of a photosensitizer, verteporfin for injection, and light exposure. The xenograft cell suspensions were injected into the dopamine-depleted striata of 40 hemiparkinsonian rats assigned to different treatment groups. Four weeks after transplantation, xenograft function (determined by methamphetamine-induced rotation) and survival (determined by immunohistochemical staining for murine neurons) were compared. Group 1 animals (xenografts pretreated with 25 ng/ml verteporfin) and Group 3 animals (no verteporfin pretreatment, but daily administration of cyclosporin A) had significantly better xenograft survival and function compared with control animals (no pretreatment with verteporfin). Group 2 animals (xenografts pretreated with 250 ng/ml verteporfin) had no significant improvement. CONCLUSIONS: This work demonstrates improved neural xenograft survival and function when using pretransplant PDT of the graft in a rodent model. The potential benefits of this new therapy are its convenience (one pretransplant treatment) and its compatibility with host immunosuppression.

Release of cytochrome c, Bax migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis.

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.

Photodynamic therapy inhibits cell adhesion without altering integrin expression.

Adhesion is a primordial cell function that, among others, regulates inflammation, metastasis, and tissue repair. To understand how these events could be affected by photodynamic therapy (PDT), we studied the effects of PDT on human foreskin fibroblast (HFF) adhesion to bovine collagen type I, human vitronectin or fibronectin. PDT, using benzoporphyrin derivative monoacid ring A (verteporfin) as the photosensitizer, inhibited cell adhesion in a drug dose-dependent manner, with no significant difference among matrices. The drug dose that killed 90% of cells within 20 h post-treatment inhibited HFF adhesion by 55%-68%. However, 45 min following PDT, a time period corresponding to that of the adhesion assay, HFF membrane integrity remained unaltered. In addition, cell surface expression of integrins was not modified for at least 2h following PDT. Western blots of cell lysates, using the anti-phosphotyrosine 4G10 monoclonal antibody, revealed that PDT prevented the adhesion-induced phosphorylation of 110-130 kDa proteins. Immunoblots of cell lysates immunoprecipitated with antibodies to focal adhesion kinase suggested that its phosphorylation was suppressed by PDT. These results demonstrate that PDT inhibits cell adhesion and affects integrin signalling without modifying cell membrane integrity or integrin expression.

Biological activities of phthalocyanines. XVII histopathologic evidence for different mechanisms of EMT-6 tumor necrosis induced by photodynamic therapy with disulfonated aluminum phthalocyanine or photofrin.

The necrosis of EMT-6 mammary murine tumors induced by photodynamic therapy (PDT) with Photofrin (PII) or disulfonated aluminum phthalocyanine (AlPcS2) was studied. Attention was given to the spontaneous evolution of angiogenesis and necrosis of such tumors in order to determine the most appropriate moment for treatment. On day 6 after tumor cell inoculation, mice were injected with photosensitizer followed by exposure to red light 24 h later, at which time optimal dye concentrations were reached in the tumor. Animals were sacrificed 3 h or 24 h after illumination and tissues were prepared for histology. Prominent cytopathic alterations were already observed at 3 h and there was massive necrosis at 24 h. In the case of PII vascular damage, congestion and hemorrhage were already present at 3 h and these changes account for the subsequent tumor necrosis through hemorrhagic infarction. With AlPcS2 these early vascular alterations were much less evident and only focal at 24 h, suggesting that AlPcS2-PDT mediated tumor necrosis involves to a large extent direct tumor cell damage.

Structure-photodynamic activity relationships of a series of 4-substituted zinc phthalocyanines.

Radioiodinated zinc phthalocyanine including [125I]ZnPcI4 and differently sulfonated [65Zn]ZnPcS (ZnPcS4, ZnPcS3, ZnPcS2 and ZnPcS1.75, a mixture of adjacent di and 25% mono) were prepared in order to study cell uptake and release kinetics in EMT-6 cells. The same compounds were evaluated for their in vitro phototoxicity and the biological parameters were compared to partition coefficients to arrive at quantitative structure-activity relationships (QSAR). At 1 microM in 1% serum, at 37 degrees C, all dyes showed rapid cell uptake during the first hour followed by a slow accumulation phase. After 24 h, the highest cellular concentration was observed with the lipophilic ZnPcI4, followed by the amphiphilic ZnPcS2 and ZnPcS1.75. The hydrophilic ZnPcS4 and ZnPcS3 showed lower uptake. Dye release from dye-loaded cells during incubation in dye-free medium could reach up to 60% and was shown to depend mainly on the amount of drug incorporated rather than the type of compound. These results suggest that care should be taken in interpreting dye toxicity data, which involve in vitro cell manipulations in dye-free medium, particularly during in vitro-in vivo protocols. The EMT-6 cell survival after 1 h or 24 h incubation with 1 microM dye in 1% serum followed by exposure to red light was assessed by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. Photocytotoxicities correlated inversely with the tendencies of the dyes to aggregate. Increased dye uptake by the cells also correlated with their activities, except for the lipophilic ZnPcI4, which showed the highest cell uptake but little phototoxicity. The QSAR between phototoxicity and the log of the partition coefficients (phosphate-buffered saline and n-octanol) gave a parabola with optimal partition values corresponding to the adjacent sulfonated ZnPcS2.

Evaluation of a 99mTc-antimyosin kit for myocardial infarct imaging.

The Fab fragment of a mouse monoclonal antibody AM(3-48) that recognizes alpha and beta-heavy chains of human atrial and ventricular myosin and beta-heavy chain of human slow skeletal muscle myosin [CardioVisionTM] was labeled with 99mTc using stannous reductant in a simple, instant kit method. The infarcted heart uptake in dogs of 99mTc-AM(3-48)Fab' was compared with that of established radiopharmaceuticals routinely used for cardiac imaging in humans. The dog infarct was induced by bringing a catheter from the femoral artery to the coronary artery where an artificial blood clot was generated. The 99mTc-AM(3-48)Fab' preparation was selectively taken up by infarcted myocardium, resulting in diagnostic quality images of the infarcted area as early as 6 hour post-injection, rendering CardioVisionTM particularly useful for SPECT imaging. Good agreement was found between the images obtained with 99mTc-Pyrophosphate and those obtained with 99mTc-AM(3-48)Fab', while the infarcted area was clearly delineated as a cold spot with 99mTc-MIBI or 201 Tl-thallous chloride. The biodistribution of 99mTc-AM(3-48)Fab' was also studied in healthy and isoproterenol-infarcted rats, from which dosimetry values in man were extrapolated. The data indicate that the kidneys will receive the highest radiation dose and that they will be the main contributors to the total radiation burden, which was estimated at 0.005 rad/mCi.

Phthalocyanine-induced photohemolysis: structure-activity relationship and the effect of fluoride.

Phthalocyanine (Pc) containing A1, Ga or Zn as central metal ligand and substituted with a varying number of sulfonic acid residues as well as additional benzene rings were synthesized and their photodynamic activity was assayed using photohemolysis of human erythrocytes as an endpoint. The Pc derivatives varied > 300-fold in their photodynamic activity. Activity correlated with binding of the dye to the cell, with the exception of some of the amphiphilic dyes where cell uptake was an order of magnitude higher than expected from the observed activity. Fluoride was shown to inhibit A1PcSn-induced photohemolysis. This effect occurred also with other A1Pc and GaPc derivatives, but the concentration of F- required to slow photohemolysis by a factor of two (Ki) varied between 4 microM and 10 mM. Fluorescence spectral studies indicated complex formation between F- and the dye, which was stronger for A1Pc than GaPc derivatives. Ultrastructural studies using scanning electron microscopy showed that the photosensitized cells were converted to spherocytes and that F- prevented this to a large extent.

Photodynamic properties of naphthosulfobenzoporphyrazines, novel asymmetric, amphiphilic phthalocyanine derivatives.

Metallo naphthosulfobenzoporphyrazines sulfonated to different degrees (M-NSBP) were prepared, and their potential as photosensitizers for the photodynamic therapy (PDT) of cancer was evaluated. M-NSBP can be viewed as hybrid molecules between sulfophthalocyanines and naphthalocyanines resulting in distinct differences in the absorption spectra between the mono-through tetrasulfonated derivatives. This feature greatly facilited their purification. Using V-79 Chinese hamster cells in vitro, the disulfonated derivatives were found slightly more photoactive than the hydrophilic trisulfonated derivatives while the monosulfonated derivative was inactive, in spite of a sixfold higher cell uptake. In the case of the di- and trisulfonated derivatives, differences in phototoxicity correlated well with their relative cell uptake. Substitution of Al for Zn had little effect on the extent of phototoxicity of the M-NSBP. In vitro PDT of the EMT-6 cells after in vivo dye administration, revealed a similar potency for direct cell killing between the di- and trisulfonated AlOH-NSBP, while the monosulfonated analog was inactive. PDT with the amphiphilic disulfonated AlOH-NSBP on the EMT-6 mammary tumor in BALB/c mice induced a significant tumor response, while the monosulfonated derivative was much less active.

Mixed acenannellated metallotetraazaporphins: a new class of amphiphilic photosensitizers for the photodynamic therapy of cancer.

The first synthesis of amphiphilic mixed acenannellated metallotetraazaporphins compounds, designed with the aim of improving cell membrane penetration, is described and the preliminary results on their photocytotoxicity are presented.