Mutagenicity of Photodynamic Therapy as Compared to UVC and Ionizing Radiation in Human and Murine Lymphoblast Cell Lines

Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (porfimer sodium) (PF) or aluminum phthalocyanine (AIPc) as the photosensitizer was determined at the thymidine kinase (TK) locus in the human lymphoblastic cell lines, TK6 and WTK1, and was compared to the mutagenicity of UVC and X-radia-tion in these cells as well as the mutagenicity of PDT in murine L5178Y lymphoblastic cell lines. Photodynamic therapy was found not to be mutagenic in TK6 cells, which possess an active p53 gene and which are relatively deficient in recombination and repair of DNA double-strand breaks. In contrast, PDT with either sensitizer was significantly mutagenic in WTK1 cells, which harbor an inactivating mutation in the p53 gene and are relatively efficient in recombination and double-strand break repair as compared to TK6 cells. The induced mutant frequency in WTK1 cells with PF as the photosensitizer was similar to that induced by UVC radiation but lower than that induced by X-radiation at equitoxic faiences/ doses. The mutant frequency induced by PDT in WTK1 cells with either photosensitizer was much lower than that induced in murine lymphoblasts at equitoxic fluences. The TK6 and WTK1 cells did not differ in their sensitivity to the cytotoxic effects of PDT, but the level of PDT-induced apoptosis was greater in TK6 than in WTK1 cells. These results indicate that the mutagenicity of PDT varies in different types of cells and may be related to the repair capabilities as well as the p53 status of the cells.     

Photophysical and Redox Properties of a Series of Phthalocyanines: Relation with Their Photodynamic Activities on TF-1 and Daudi Leukemic Cells

Abstract— The photodynamic therapy (PDT) efficiency of five phthalocyanines, chloroaluminum phthalocyanine (AlPc), dichlorosilicon phthalocyanine (SiPc), bis (tri- n -hexylsi-loxy)silicon phthalocyanine (PcHEX), bis (triphenyl-siloxy)silicon phthalocyanine (PcPHE) and nickel phthalocyanine (NiPc), was assessed on two leukemic cell lines TF-1 and erythroieukemic and B lymphoblastic cell lines, Daudi, respectively. AlPc showed the best photocytotox-icity leading to 0.008 surviving fraction at 2 × 10⁻⁹ M for TF-1 and 4 × 10⁻⁹ M for Daudi. At 5 × 10⁻⁷ M , SiPc and PcHEX induced a significant photokilling, whereas NiPc and PcPHE were inactive. Laser flash photolysis and photoredox properties of the phthalocyanines were investigated to try to relate these parameters with the biological effects. AlPc showed the longest triplet lifetime: 484 fis in dimethyl sulfoxide/H₂O. This value was increased up to 820 u.s when AlPc was complexed with human serum albumin used as a membrane model. Such an enhancement was not observed with the silicon phthalocyanines. Upon irradiation, all the phthalocyanines generated singlet oxygen with 0.29–0.37 quantum yield values. The reduction potentials of the excited states obtained from measurement in the ground state and energy of the excited triplets show that AlPc is the best electron acceptor. The in vitro photocytotoxicity observed and the measured parameters are in agreement with a key role of electron transfer in PDT assays involving these phthalocyanines.     

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.     

Apoptosis is an Early Event During Phthalocyanine Photodynamic Therapy-Induced Ablation of Chemically Induced Squamous Papillomas in Mouse Skin

Abstract— Photodynamic therapy (PDT) is a promising new modality to treat malignant neoplasms including superficial skin cancers. In our search for an ideal photosensitizer for PDT, Pc 4, a silicon phthalocyanine, has shown promising results both in in vitro assays and in implanted tumors. In this study we assessed the efficacy of Pc 4 PDT in the ablation of murine skin tumors; and the evidence for apoptosis during tumor ablation was also obtained. The Pc 4 was administered through tail vein injection to SENCAR mice bearing chemically induced squamous papillomas, and 24 h later the lesions were illuminated with an argon ion-pumped dye laser tuned at 675 nm for a total light dose of 135 J/cm². Within 72-96 h, almost complete tumor shrinkage occurred; no tumor regrowth was observed up to 90 days post-PDT. As evident by nucleosome-size DNA fragmentation, appearance of apoptotic bodies in hematoxylin and eosin staining and direct immunoperoxidase detection of digoxigenin-labeled genomic DNA in sections, apoptosis was clearly evident 6 h post-PDT at which time tumor shrinkage was less than 30%. The apoptotic bodies, as evident by the condensation of chromatin material around the periphery of the nucleus and increased vacuolization of the cytoplasm, were also observed in electron microscopic studies of the tumor tissues following Pc 4 PDT. The extent of apoptosis was greater at 15 h than at 6 and 10 h post-PDT. Taken together, our results clearly show that Pc 4 may be an effective photosensitizer for PDT of nonmelanoma skin cancer, and that apoptosis is an early event during this process.     

Photodynamic inactivation of retroviruses by phthalocyanines: the effects of sulphonation, metal ligand and fluoride.

The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.     

Photodynamic Therapy: Tumor Targeting with Adenoviral Proteins

A brief summary of the mechanisms involved in photodynamic therapy (PDT) and the role of delivery vehicles for photosensitizer targeting is addressed. Phthalocyanines (Pc) have been coupled to adenovirus type 2 capsid proteins including the hexon, the penton base and the fiber to enhance their target selectivity. Adenovirus penton base proteins contain the arginine-glycine-aspartic acid peptidic sequence (RGD) motif known to bind with great affinity and high specificity to integrin receptors, expressed by several types of cancer. Tetrasulfonated aluminum phthalocyanine (AlPcS4) was covalently coupled to the various capsid proteins via one or two caproic acid spacer chains (A1 or A2) in 7:1 up to 66:1 molar ratios. The capacity of the bioconjugates for singlet oxygen production, as measured by an L-tryptophan oxidation assay, was strongly reduced, likely reflecting scavenging by the carrier. Cell adsorption and in vitro photocytotoxicity assays were carried out using the A549 and HEp2 human cell lines expressing integrin receptors, and one murine, the EMT-6 cell line, which lacks receptors for the RGD sequence. The AlPcS4A2-protein complexes induced greater cytotoxicity as compared to the analogous AlPcS4A1 preparations. The penton base-AlPcS4A2 derivative was the more phototoxic for all cell lines tested. Tumor response studies using Balb/c mice with EMT-6 tumor implants demonstrated that the free AlPcS4A2 induced complete tumor regression at a dose of 1 mumol/kg and 400 J/cm2, which is comparable to the activity of the known AlPcS2adj. A mixture of adenovirus type 2 soluble proteins covalently labeled with AlPcS4A2 required 0.5 mumol/kg to induce the same response with the same light dose, suggesting that the high affinity RGD/receptor complex is able to target Pc for PDT.     

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.     

Immunosuppressive Effects of Silicon Phthalocyanine Photodynamic Therapy

The purpose of this study was to determine if silicon phthalocyanine 4 (Pc 4), a second-generation photosensitizer being evaluated for the photodynamic therapy (PDT) of solid tumors, was immunosuppressive. Mice treated with Pc 4 PDT 3 days before dinitrofluorobenzene sensitization showed significant suppression of their cell-mediated immune response when compared to mice that were not exposed to PDT. The response was dose dependent, required both Pc 4 and light and occurred at a skin site remote from that exposed to the laser. The immunosuppression could not be reversed by in vivo pre-treatment of mice with antibodies to tumor necrosis factor-alpha or interleukin-10. These results provide evidence that induction of cell-mediated immunity is suppressed after Pc 4 PDT. Strategies that prevent PDT-mediated immunosuppression may therefore enhance the efficacy of this therapeutic modality.     

A Requirement for Bid for Induction of Apoptosis by Photodynamic Therapy with a Lysosome- but Not a Mitochondrion-targeted Photosensitizer

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid^−/− cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.     

Rapid Tyrosine Phosphorylation of HS1 in the Response of Mouse Lymphoma L5178Y-R Cells to Photodynamic Treatment Sensitized by the Phthalocyanine Pc 4

Abstract— The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular signal transduction pathways in murine lymphoma L5178Y-R cells has been assessed by observing increases in protein tyrosine phosphorylation at early times post-PDT. Western blot analysis with an anti-phosphotyrosine antibody revealed a dramatic increase in phosphorylation of two major protein bands of Mr -80000 and -55000 in response to PDT. The increase was PDT dose-dependent, occurred as early as 20 s after initiation of light exposure of Pc 4-pre-loaded cells and was amplified by the presence of the protein tyrosine phosphatase inhibitor, sodium ortho-vanadate (NaV0₄). By immunoprecipitation, one of the Mr –80000 phosphorylated proteins has been identified as HS1, a substrate of nonreceptor-type protein tyrosine kinases. Although vanadate greatly enhanced the level and extent of PDT-induced phosphorylation, it had no influence on overall photocytotoxicity or on the rate of apoptotic DNA fragmentation. Genistein, an inhibitor of protein tyrosine kinases, diminished tyrosine phosphorylation of the Mr –80000 and other proteins and dramatically potentiated cell killing induced by PDT but did not significantly affect PDT-induced apoptosis. The results suggest that PDT rapidly activates a membrane-associated src family kinase(s) in L5178Y-R cells, one substrate of which is HS1, and that protein tyrosine phosphorylation is part of a stress response, protecting a portion of the cells from the lethal effects of PDT but not altering the mechanism by which they die.     

The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg⁻¹ Pc 4 iv only, laser irradiation (150 J cm⁻²) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro , decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT ( R ² = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.     

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.     

Association of Ceramide Accumulation with Photodynamic Treatment-Induced Cell Death

Abstract— Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitiz-er Pc 4 (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome-size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotox-icity.     

APOPTOSIS DURING PHOTODYNAMIC THERAPY-INDUCED ABLATION OF RIF-1 TUMORS IN C3H MICE: ELECTRON MICROSCOPIC, HISTOPATHOLOGIC AND BIOCHEMICAL EVIDENCE

Abstract Very little is known about the applicability of the metabolic and biochemical events observed in cell culture systems to in vivo tumor shrinkage following photodynamic therapy (PDT). The purpose of this study was to assess whether PDT induces apoptosis during tumor ablation in vivo . We treated radiation-induced fibrosarcoma (RIF-1) tumors grown in C3H/HeN mice with PDT employing three photosensitizers, Photofrin-II, chloroaluminum phthalocyanine tetrasulfonate, or Pc IV (a promising phthalocyanine developed in this laboratory). Each photosensitizer was injected intraperitoneally and 24 h later the tumors were irradiated with an appropriate wavelength of red light using an argon-pumped dye laser. During the course of tumor shrinkage, the tumors were removed at 1, 2, 4 and 10 h post-PDT for DNA fragmentation, histopathologic, and electron microscopic studies. Markers of apoptosis, viz . the ladder of nucleosome-size DNA fragments, increased apoptotic bodies, and condensation of chromatin material around the periphery of the nucleus, were evident in tumor tissue even 1 h post-PDT; the extent of these changes increased during the later stages of tumor ablation. No changes were observed in tumors given photosensitizer alone or irradiation alone. Our data suggest that the damage produced by in vivo PDT may activate endonucleolysis and chromatin condensation, and that apoptosis is an early event in tumor shrinkage following PDT.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

Hypericum perforatum L. extract - novel photosensitizer against human bladder cancer cells

The polar methanolic fraction (PMF) of the Hypericum perforatum L. extract has recently been developed and tested as a novel, natural photosensitizer for use in the photodynamic therapy (PDT), and photodynamic diagnosis (PDD). PMF has been tested on HL-60 leukemic cells and cord blood hemopoietic progenitors. In the present study, the efficacy of PMF as a phototoxic agent against urinary bladder carcinoma has been studied using the T24 (high grade metastatic cancer), and RT4 (primary low grade papillary transitional cell carcinoma) human bladder cancer cells. Following cell culture incubation, PMF was excited using 630 nm laser light. The photosensitizer exhibited significant photocytotoxicity in both cell lines at a concentration of 60microg/ml, with 4-8 J/cm(2) light dose, resulting in cell destruction from 80% to 86%. At the concentration of 20microg/ml PMF was not active in either cell line. These results were compared with the results obtained in the same cell lines, under the same conditions with a clinically approved photosensitizer, Photofrin. Photofrin was used in the maximum clinically tolerable dose of 4microg/ml, and it was also excited with 630 nm laser light. In the T24 cell Photofrin exhibited slightly less photocytotocixity, compared with PMF, resulting in 77% cell death with 8J/cm(2) light dose. However, against the RT4 cells Photofrin resulted in minimal cell death (9%) with even 8J/cm(2) light dose. Finally, the type of cell death induced by PMF photoactivation was studied using flow cytometry and DNA laddering. Cell death by PMF photodynamic action in these two bladder cell lines is caused predominently by apoptosis. The reported significant photocytotoxicity, selective localization, natural abundance, easy, and inexpensive preparation, underscore that the PMF extract hold the promise of being a novel, effective PDT photosensitizer.     

Semiconductor quantum dots for photodynamic therapy

The applicability of semiconductor QDs in photodynamic therapy (PDT) was evaluated by studying the interaction between CdSe QDs with a known silicon phthalocyanine PDT photosensitizer, Pc4. The study revealed that the QDs could be used to sensitize the PDT agent through a fluorescence resonance energy transfer (FRET) mechanism, or interact directly with molecular oxygen via a triplet energy-transfer process (TET). Both mechanisms result in the generation of reactive singlet oxygen species that can be used for PDT cancer therapy.     

Phthalocyanine 4 (Pc 4) photodynamic therapy of human OVCAR-3 tumor xenografts

Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin has been approved by the U.S. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc-OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR-3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR-3 tumors (approximately 80-130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (lambda = 672 nm, 150 J/cm2, 150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post-PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post-PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post-PDT. Western blotting showed the cleavage of the 116 kDa native poly(ADP-ribose) polymerase (PARP) into fragments of approximately 90 kDa, another indication of apoptosis, and the presence of p21/WAF1/CIP1 (p21) in all PDT-treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4-PDT response. Clinical trials of Pc 4-PDT are warranted.     

LARGE MUTAGENIC LESIONS ARE INDUCED BY PHOTODYNAMIC THERAPY IN MURINE L5178Y LYMPHOBLASTS*

Abstract— Mutagenic lesions at the thymidine kinase locus (tk) in mouse lymphoma L5178Y (LY) cells treated with red light and either Photofrin (PF) or chloroaluminurn phthalocyanine (AIPc) as the photosensitizer were compared in the relatively photodynamic therapy (PDT)-sensitive strain LY-R16 and the relatively resistant strains LY-S1 and LY-SR1. Southern blot analysis revealed that 92% (36/39) of the PDT-induced thymidine kinase (TK ^?/-) mutants of strains LY-R16 and LY-SR1 lost the entire active tk allele. (Strain LY-S1 lacks a known tk polymorphism and has not been analyzed for loss of the active tk allele.) A decrease in galactokinase (GK) activity in the TK^?/- mutants has been taken as an indication that the mutagenic lesion extends from the tk gene to the closely linked galactokinase gene (gk). Using PF as the photosensitizer, GK activity was decreased in 45% of the LY-R16 mutants and in 22% of the LY-S1 and LY-SR1 mutants. With photoactivated AIPc, 59% of the TK ^?/- mutants of strains LY-S1 and LY-SR1 showed GK inactivation. (LY-R16 mutants were not analyzed because of the low LY-R16 mutant frequency induced by PDT with AlPc.) Thus, many of the TK^?/- mutants of LY cells induced by PDT with either PF or AlPc harbor multilocus lesions.     

The immunosuppressive effects of phthalocyanine photodynamic therapy in mice are mediated by CD4+ and CD8+ T cells and can be adoptively transferred to naive recipients

Photodynamic therapy (PDT) is a promising treatment modality for malignant tumors but it is also immunosuppressive which may reduce its therapeutic efficacy. The purpose of our study was to elucidate the role of CD4+ and CD8+ T cells in PDT immunosuppression. Using silicon phthalocyanine 4 (Pc4) as photosensitizer, nontumor-bearing CD4 knockout (CD4-/-) mice and their wild type (WT) counterparts were subjected to Pc4-PDT in a manner identical to that used for tumor regression (1 cm spot size, 0.5 mg kg(-1) Pc4, 110 J cm(-2) light) to assess the effect of Pc4-PDT on cell-mediated immunity. There was a decrease in immunosuppression in CD4-/- mice compared with WT mice. We next examined the role of CD8+ T cells in Pc4-PDT-induced immunosuppression using CD8-/- mice following the same treatment regimen used for CD4-/- mice. Similar to CD4-/- mice, CD8-/- mice exhibited less immunosuppression than WT mice. Pc4-PDT-induced immunosuppression could be adoptively transferred with spleen cells from Pc4-PDT treated donor mice to syngenic naive recipients (P < 0.05) and was mediated primarily by T cells, although macrophages were also found to play a role. Procedures that limit PDT-induced immunosuppression but do not affect PDT-induced regression of tumors may prove superior to PDT alone in promoting long-term antitumor responses.