Search

Photodynamic therapy (PDT), which uses a light-sensitive compound and laser irradiation, is a light-based oncological treatment modality. PDT offers an alternative, less invasive treatment for various malignant tumors, such as esophageal cancer (EC), through a photochemical reaction induced by photofrin-II or other oncotropic photosensitizers without severe complications. Previous studies has shown that cancerous tissues accumulated more photosensitizers than paired normal tissues, however, whether it is cellular or vascular mechanisms remains unknown. Herein, in vivo and in vitro examinations were performed to study the mechanisms by which photofrin-II effectively and specifically killed EC cells. In this study, EC tissue of patients treated with photofrin-II, human ESCC cellline SHEEC and parental normal cellline SHEE, primary culture cells of EC tissue were used. The concentration of photofrin-II in cells were evaluated by high-performance liquid chromatography (HPLC). The results exhibited that accumulation of photofrin-II in cancerous cells were significantly higher than that in non-cancerous cells (p<0.05) under certain dose and time period of incubation of photofrin-II. In summary, our study showed that, photofrin-II specifically accumulated in EC cells in vivo and in vitro after controlling for vascular factors, which provided strong evidence that maybe the cellular factor is the main mechanism by which photofrin-II-mediated PDT selectively caused EC cells death., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Despite initially positive responses, recurrences after Photodynamic treatment (PDT) can occur and there is need for improvement in the effectiveness of PDT. Our study uniquely showed that there was a significantly gap junctional intercellular communication (GJIC)-dependent PDT cytotoxicity. The presence of GJIC composed of Connexin 32 increased the PDT phototoxicity in transfected HeLa cells and in the xenograft tumors, and the enhanced phototoxicity of Photofrin-mediated PDT by GJIC was related with ROS and calcium pathways. Our study indicates the possibility that up-regulation or maintenance of gap junction functionality may be used to increase the efficacy of PDT. The phototoxicity effect of Photofrin was substantially greater in Dox-treated cells, which expressed the Cx32 and formed the GJ, than Dox-untreated., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53(+/+)) and H1299 (p53(-/-)) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

Photodynamic therapy has been considered ineffective for melanomas because of the competition between the absorbance of melanin from the melanoma and the absorbance of photosensitizers at the photosensitizer excitation light wavelength. Melanomas show considerable heterogeneity and resistance to phototherapy. The effectiveness of photodynamic therapy could be intensified by electroporation for enhanced transport of a photosensitizer by transient pores in the membrane. In this study, photodynamic therapy combined with electroporation was tested in vitro on the human melanoma cell lines melanotic melanoma (MeWo) and amelanotic melanoma (C32). Control experiments were conducted on human keratinocytes (HaCaT). Photofrin was used as a photosensitizer. Photosensitizer distribution, cloning efficacy test, comet assay, and assessment of apoptotic proteins were performed. Melanin levels were determined before and after photodynamic therapy. The experiments indicated that electroporation effectively supports the photodynamic method. It was found that photodynamic therapy with electroporation efficiently induces apoptosis in melanotic and amelanotic melanoma cells.

The influence of electroporation on the Photofrin uptake and distribution was evaluated in the breast adenocarcinoma cells (MCF-7) and normal Chinese hamster ovary cells (CHO) lacking voltage-dependent channels in vitro. Photofrin was used at a concentration of 5 and 25 μM. The uptake of Photofrin was assessed using flow cytometry and fluorescence microscopy methods. Cells viability was evaluated with crystal violet assay. Our results indicated that electropermeabilization of cells, in the presence of Photofrin, increased the uptake of the photosensitizer. Even at the lowest electric field intensity (700 V/cm) Photofrin transport was enhanced. Flow cytometry results for MCF-7 cells revealed ~1.7 times stronger fluorescence emission intensity for cells exposed to Photofrin and electric field of 700 V/cm than cells treated with Photofrin alone. Photofrin was effective only when irradiated with blue light. Our studies on combination of photodynamic reaction with electroporation suggested improved effectiveness of the treatment and showed intracellular distribution of Photofrin. This approach may be attractive for cancer treatment as enhanced cellular uptake of Photofrin in MCF-7 cells can help to reduce effective dose of the photosensitizer and exposure time in this type of cancer, diminishing side effects of the therapy.

Objective: Our final goal is to develop an appropriate method using nuclear medicine technique for monitoring the effect and prediction of Photodynamic Therapy (PDT) on tumors. The aim of this study is to evaluate the effect of PDT on tumor cells in vitro using (18)F-FDG and (99m)Tc-MIBI as tracers., Methods: Five tumor cell lines (A431, DU145, H1650, LS180, SHIN3) with varied characteristics were irradiated after incubating for 24 h with several doses of Photofrin (PF). Singlet oxygen was monitored by the near-IR emission detection system during irradiation and generated (1)O2 was calculated. PDT effects were rapidly evaluated by nuclear medicine techniques (uptake of (18)F-FDG and (99m)Tc-MIBI) and traditional methods for cell viability (MTT and trypan blue assays) at 3 h after PDT. Intracellular PF concentration was measured by absorption spectrometer and cell protein content was measured by the Lowry method. (18)F-FDG uptake, (99m)Tc-MIBI uptake, singlet oxygen, and intracellular PF concentration were standardized by protein content. Decrease % of (18)F-FDG and (99m)Tc-MIBI, MTT, and trypan blue was normalized to the control group., Results: Decrease % of (18)F-FDG was exponentially related to decrease % of MTT (R (2) = 0.650, P < 0.01) while decrease % of (99m)Tc-MIBI was linearly related to that of MTT (R (2) = 0.719, P < 0.01). The decrease % of MTT was more sensitive than that of trypan blue. However, neither (1)O2 nor PF uptake was correlated with sensitivity to PDT. In addition, (18)F-FDG uptake before PDT was linearly related to decrease % of MTT (R (2) = 0.800, P < 0.05)., Conclusions: Our findings in in vitro studies suggest that (99m)Tc-MIBI is better than (18)F-FDG for early evaluation of PDT effect, but (18)F-FDG uptake may be used to predict PDT sensitivity before therapy.

Despite remarkable progress in diagnosis and treatment, malignant glioma, a highly lethal cancer of the central nervous system, remains incurable. Although glioma stem-like cells (GSCs) represent a relatively small fraction of the cells in malignant glioma, they can proliferate and self renew extensively, being crucial for tumor recurrence. Cancer treatment by sonodynamic therapy (SDT) chiefly depends on antitumor effects of reactive oxygen species (ROS) generated from a sonosensitizer activated by ultrasound. Although SDT effectively kills glioma cells, its efficiency against GSCs is not established. We attempted to compare the susceptibility of GSCs to SDT, using Photofrin, a porphyrin-derivative photosensitizer, with that of glioma cells. Cell viability and apoptosis assays showed that SDT damaged both GSCs and U251 glioma cells, but GSCs were significantly less susceptible to SDT (p < 0.01). To elucidate the mechanism of the antitumor effects of SDT, we evaluated intracellular ROS production and Photofrin uptake: ROS production and Photofrin content were significantly lower (p < 0.01) in GSCs than in U251 glioma cells. Thus, cellular differences in sonosensitizer uptake and ROS production influence the antitumor effects of SDT. Furthermore, the resistance of GSCs may be caused by decreased sonosensitizer uptake due to ABCG2 overexpression.

Background: Melanoma is the most severe of skin neoplasms as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens up new prospects in the treatment of this tumor. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cellular and molecular mechanisms which mediate oxidative stress in cells., Objectives: The aim of this study was to evaluate in vitro the influence of photodynamic therapy on the human melanoma Me45 cell line., Material and Methods: Photofrin (Ph) was used as a photosensitizer., Results: Viability studies have shown that there are significant differences between cells after PDT and cells without irradiation. After 24 hours of incubation with a 20 microg/ml concentration of Ph and with irradiation, less than 20% of the cells survived. In the control (without PDT), 65% of the cells survived., Conclusions: The mitochondrial localization of Ph is significant, as it may lead to disturbances of mitochondrial transmembrane potential and finally to apoptotic cell death. The expressions of manganese superoxide dismutase and heme oxygenase and the level of carbonyl and thiol groups are indicating factors for oxidative stress in Me45 cells.

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT), the concept of cancer treatment through the selective uptake of a light-sensitive agent followed by exposure to a specific wavelength, is limited by the transport of a photosensitizer (PS) to the tumor tissue. Porphyrin, an important PS class, can be used in PDT in the form of its prodrug molecule 5-aminolevulinic acid (5-ALA). Unfortunately, its poor pharmacokinetic properties make this compound difficult to administer. Two different methods for eliminating this problem can be distinguished. The first approach is to play with its formulation in order to improve the drug's applicability. The second approach, which is to find possible 5- ALA prodrugs, is an example of the double-prodrug method, a strategy often used in modern drug design. In this approach, the biological mechanisms in a long biosynthetic pathway involving several steps must be completed before the active drug appears. Recently, an idea of enhancing PDT sensitization using the so-called iron chelators seemed to increase the accumulation of protoporphyrin in cells. At the same time, iron chelators can destroy tumor cells by producing active oxygen after the formation of an active drug by chelating iron in the cancer cells. Thus, in the latter case, the therapy resembles a prodrug strategy. The mechanism can be explained by the Fenton reaction. Vitamin C is another example of a potential anticancer agent of this type.

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by exposure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant colon cancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulation of photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indicate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity after PDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidative stress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy of PDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation of photosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT.

Compared to current treatments including surgery, radiation therapy, and chemotherapy, PDT offers the advantage of an effective and selective method of destroying diseased tissues without damaging surrounding healthy tissues. One of the aspects of antitumour effectiveness of PDT is related to the distribution of photosensitizing drugs. The localization of photosensitizers in cytoplasmic organelles during PDT plays a major role in the cell destruction; therefore, intracellular localization of Ph in malignant and normal cells was investigated. The cell lines used throughout the study were: human malignant A549, MCF-7, Me45 and normal endothelial cell line HUV-EC-C. After incubation with Ph cells were examined using fluorescence and confocal microscopy to visualize the photosensitizer accumulation. For cytoplasm and mitochondria identification, cells were stained with CellTracker Green and MitoTracker Green, respectively. Distribution of Ph was different in malignant and normal cells and dependent on the incubation time. The maximal concentration of Ph in two malignant cell lines (A549 and MCF-7) was observed after 4 hours of incubation, and the most intensive signal was observed around the nuclear envelope. Intracellular distribution of Ph in the Me45 cell line showed that the fluorescence emitted by Ph overlaid that from MitoTracker. This indicates preferential accumulation of the sensitizer in mitochondria. Our results based on the mitochondrial localization support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of >/=3 microg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.

Purpose: The aim of the present study was to potentiate the antitumor effectiveness of photodynamic therapy (PDT). A cDNA microarray analysis was used to evaluate the gene expression pattern after Photofrin-mediated PDT to find more effective combination treatment with PDT and inhibitor(s) of the identified gene product(s) overexpressed in tumor cells., Experimental Design: Atlas Mouse Stress Array was used to compare the expression profile of control and PDT-treated C-26 cells. The microarray results have been confirmed using Western blotting. Cytostatic/cytotoxic in vitro assay as well as in vivo tumor models were used to investigate the antitumor effectiveness of PDT in combination with cyclooxygenase (COX) 2 inhibitors., Results: PDT induced the expression of 5 of 140 stress-related genes. One of these genes encodes for COX-2, an enzyme important in the tumor progression. Inhibition of COX-2 in vitro with NS-398, rofecoxib, or nimesulide, or before PDT with nimesulide did not influence the therapeutic efficacy of the treatment. Administration of a selective COX-2 inhibitor after PDT produced potentiated antitumor effects leading to complete responses in the majority of treated animals., Conclusions: COX-2 inhibitors do not sensitize tumor cells to PDT-mediated killing. However, these drugs can be used to potentiate the antitumor effectiveness of this treatment regimen when administered after tumor illumination.

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.

The pharmacological modulation of the uptake of porphyrin derivatives in cultured C6 glioma cells was investigated by means of spectrofluorometric analysis both in single cells and in cell homogenates. The influence of drugs acting as beta-receptor agonists or antagonists was studied in cells grown to semiconfluency. Isoproterenol (ISO), a beta-receptor agonist, enhanced the intracellular fluorescence intensity of both Photofrin and protoporphyrin IX (PpIX). A treatment with a beta-receptor antagonist I-propranolol (PRO), simultaneous with ISO, resulted in an intracellular Photofrin fluorescence signal comparable to that of the control cells, indicating the specificity of the pharmacological action. The pharmacological treatment seemed particularly effective with the aggregated species. This is suggested by the relative increase of the band at 670 nm, being greater than that in the 630 nm band in the emission spectra of Photofrin and PpIX, and by the comparison of the fluorescence intensity on cell homogenates measured both in the absence and in the presence of cetyltrimethyl-ammonium bromide as a detergent.

The binding of monomeric (Hp) and oligomeric (PHE) forms of porphyrin to glioblastoma (U-87MG) cells and the photosensitization of these cells have been studied. Upon binding to U-87MG cells, Hp and PHE exhibited fluorescence bands at 615 and 636 nm, respectively. The fluorescence and absorption spectra of Hp, HpD and PHE, measured in different solvents, suggest that the 615 nm band may arise due to the binding of monomeric as well as aggregated forms of porphyrin to the hydrophilic sites in the cells whereas the 636 nm band may be due to the binding of an aggregated form of porphyrin to the hydrophobic sites. The photosensitivity of cells and photo-induced lipid peroxidation were measured as a function of light dose. Cells were found most photosensitive to PHE followed by HpD and Hp. The photosensitivity of cells correlates well with the fluorescence intensity of cell bound dye at 636 nm. These results suggest that the binding of the oligomeric component of HpD to hydrophobic sites in the cells is responsible for the enhancement in the photosensitivity.

Photofrin, a photosensitizer used in the photodynamic therapy of cancer, selectively localizes in cellular membranes. Upon exposure to visible light, Photofrin produces singlet oxygen (1O2), which reacts with membrane polyunsaturated fatty acids forming lipid hydroperoxides. Transition metals, such as Fe2+, catalyze the production of cytotoxic free radicals from lipid hydroperoxides. Ascorbate reduces ferric to ferrous iron, further augmenting lipid peroxidation. Therefore, to increase the efficacy of Photofrin photosensitization, we added 20 microM ferrous sulfate and 100 microM ascorbic acid, in an aqueous layer over SCC-25 oral squamous cell carcinoma cells during in vitro illumination. In electron paramagnetic resonance spin trapping experiments, using POBN (alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone), we observed that the presence of this pro-oxidant combination greatly increases the production of membrane-derived lipid free radicals. The effect was time dependent but only partially concentration dependent. Trypan blue dye exclusion demonstrated that this increase in lipid radical formation correlated with cytotoxicity. These observations support the hypothesis that Photofrin photosensitization leads to lipid hydroperoxide formation, which increases the cell's susceptibility to iron-induced Fenton chemistry. The resulting free radical-mediated lipid peroxidation results in cell death. From these data we hypothesize that the efficacy of photodynamic therapy of superficial cancer might be increased by the topical application of the pro-oxidant combination of iron and ascorbate. Furthermore, their use will probably allow lower doses of Photofrin without compromising antitumor effect.

Human serum albumin (HSA) is one of the key components in human blood that may influence drug distribution. As such, it is important to know the affinity of any drug for albumin. Previously, Photofrin, a mixture of monomeric, dimeric and oligomeric porphyrins, has been subjected to HSA binding studies. However, due to its complex nature, binding studies on Photofrin or other hematoporphyrin derivatives with HSA are inconclusive. In this report, the binding properties of some components (dimers and trimers) of Photofrin and the relationship between murine photosensitizing efficacy and those binding properties were investigated. The interaction of these porphyrins with HSA was investigated by direct ultrafiltration and fluorescent titration techniques with fluorescent probes such as dansyl-L-proline (DP), which is known to interact selectively with site II on HSA. Porphyrins also were tested for antitumor activity in a mouse model following intravenous administration and exposure to laser light. Together, the results suggest that the photosensitizers that were preferentially bound to site II of HSA were most effective at controlling murine tumor regrowth.

Unilamellar suspensions of dimyristoylphosphatidylcholine (DMPC) can be utilized to remove Photofrin from the erythrocyte. This enables correlation of the Photofrin membrane-binding processes with Photofrin-sensitized photolysis. The observed rates of erythrocyte biding as well as the observed rates of removal of PHotofrin from the erythrocyte membrane suggest the existence of two Photofrin species that differ in their rates of exchange between the erythrocyte and buffer phases. Selective depletion and readdition of these Photofrin species to the erythrocyte membrane permits evaluation of their separate and joint photolytic efficiencies. These rapidly and slowly exchanging membrane-bound Photofrin species are separately much less efficient photosensitizers than the two species together. The two Photofrin species exhibit essentially identical fluorescence emission spectra in the presence of DMPC. Nevertheless, models consistent with the results involve partitioning by chemically distinct Photofrin components or partitioning of chemically similar Photofrin components into distinct membrane environments, or a combination of these.

In an attempt to identify novel compounds useful for the optimization of Photodynamic Therapy (PDT), the tissue localization of new synthetic porphyrins was compared with Photofrin II in nude mice xenografted with a human small cell lung cancer (POVD). Three haematoporphyrin analogues were selected for this study based on prior in vitro photosensitivity screening of a series of 15 such derivatives, as well as on the basis of improved localization in C6 gliomas in mice. Two of the porphyrins yielded better tumour:normal lung ratios than Photofrin II and, of these two, one (P13) is known to exhibit good photosensitization properties both in vitro and in vivo, and is therefore a good candidate as a lead compound for the development of porphyrins suitable for the photodynamic treatment of lung tumours.

We have examined the effectiveness of photodynamic therapy against R3230AC rat mammary adenocarcinoma and human mesothelioma as xenografts in the same host. The results demonstrate that the xenografted human tumour is significantly more responsive to photodynamic treatment than the rodent mammary tumour. Studies also showed that the mesothelioma xenograft was fluence rate- and fluence-dependent while the rat tumour exposed to the same conditions demonstrated neither of these dependencies. This disparity in response was not attributable to a difference in either whole-tumour uptake or subcellular distribution of the porphyrin photosensitiser. Analysis of the effects of visible irradiation on cytochrome c oxidase activity, measured in mitochondria prepared from tumours borne on hosts injected with photosensitiser, demonstrated that photoradiation-induced enzyme inhibition was significantly greater in mesothelioma than in R3230AC mammary tumour preparations. However, in parallel studies conducted in vitro, when photosensitiser and light were delivered to previously unperturbed mitochondria, rates of enzyme inhibition were not significantly different. Both tumours were established in long-term cell culture. While the uptake of porphyrin photosensitiser was equivalent in both cell lines, the R3230AC cells displayed a significantly greater photosensitivity than the mesothelioma cells. The data presented here demonstrate that the mechanisms that govern response to photodynamic therapy are complex, but in the case of these two xenografted tumours host response to therapy is not likely to play a significant role.

Photodynamic therapy using i.v. injected porphyrin photosensitizers have been used to treat selected cases of superficial bladder cancer. Since cutaneous photosensitivity, lasting 6-8 weeks, is a well known undesirable side effect of this therapy, we instilled the photosensitizers intravesically in rats and compared the uptake of photosensitizers in different tissues by this route of administration with the uptake after intravenous injection. The intravesical mode of delivery enhanced photosensitizer uptake in the bladder wall, while giving low concentrations in extravesical organs. Intravesical instillation of the photosensitizers may therefore increase their efficacy and reduce phototoxicity as compared with intravenous injection. Comparing the results obtained by two assays, one based on porphyrin fluorescence and the other based on the application of radioactively labelled photosensitizers, it was concluded that the i.v. administration route may result in tissue uptake of significant amounts of aggregated non-fluorescent, supposedly inactive drug, while the intravesical administration led to less uptake of aggregates relative to active drug monomers.

The capability of human promyelocytic leukemia cells HL60 to be induced to differentiate to various stages along the monocytic or myelocytic pathway was exploited for investigation of the uptake of selected photosensitizers by diverse types of cells of the same origin. The results showed that there was no substantial difference in photofrin uptake between noninduced HL60 cells, immature monocytes, immature neutrophils and cells differentiated along the eosinophilic pathway. In contrast, HL60 cells differentiated into macrophages (HL60 phi) exhibited markedly increased photofrin uptake, which was further enhanced by their pretreatment with bacterial lipopolysaccharide. Similar results were obtained with other photosensitizers tested: di- and tetrasulfonated aluminum phthalocyanines (A1PcS2 and A1PcS4), tetrasulfonated zinc phthalocyanine (ZnPcS4), tetraphenylporphine tetrasulfonate (TPPS4) and benzoporphyrin derivative monoacid (BPD). Despite marked differences in the state of self-aggregation and other chemical properties of these compounds, the degree of their preferential uptake by HL60 phi cells showed very little variation. In a typical experiment, the uptake of these photosensitizers by HL60 phi cells was four to five times higher than the uptake by noninduced HL60 cells. In addition to the fluorometric assay employed in most of the experiments, cellular concentration of A1PcS4 was determined by measurement of elementary aluminum using atomic absorption spectroscopy.

A 24 h preculture of HT29-18 human colonic adenocarcinoma cells with the sterol synthesis inhibitor lovastatin at concentrations of 0.1-0.5 microM markedly increased the photocytotoxic effect of photofrin II delivered to cells by low density lipoproteins. Under the same conditions, LDL binding and photofrin II (PII) uptake by HT29 cells increased about 1.8-fold and 1.5-fold, respectively. These results suggest that hydroxymethylglutaryl-coenzyme A reductase inhibitors could be useful for potentiating the photodynamic therapy of tumors by PII.

Photodynamic effects on the nuclear envelope of human skin fibroblasts were investigated by confocal laser fluorescence microscopy and transmembrane resting potential measurements. The results show staining of the nuclear envelope after short incubation times with Photosan III, Photofrin II and haematoporphyrin derivative (HPD) enriched with monomers. Maximum staining was found at the centre of the nuclear envelope. The sequence of fluorescence intensity was HPD enriched with monomers > Photofrin II > Photosan III. After lethal treatment with Photosan III and tetrasulphonated aluminium chloride phthalocyanine, the nuclear transmembrane potential of the nuclear membrane decreased from -20 mV to about -10 mV with reference to the plasma membrane potential.

Predicting the effects of photodynamic therapy (PDT) and Nd: YAG laser coagulation requires knowledge of the light distribution in tumor and surrounding tissue. Therefore, absorption and scattering coefficients and the average cosine of the scattering angle (the anisotropy factor) were measured in rat liver and tumor at 632.8 and 1064 nm. A syngenic colon adenocarcinoma CC531 was implanted subcutaneously in two groups of 7 Wag/Rij rats. In one group Photofrin was administered 5 mg/kg intravenously 48 h before determination of optical properties. Two months after inoculation, samples were taken from tumor and liver and optical properties determined using indirect methods with two integrating spheres. The absorption coefficient was larger in liver than in tumor at 632.8 nm (P < 0.0005), whereas the scattering coefficient was larger in tumor than in liver at 1,064 nm (P < 0.05). Addition of Photofrin increased the scattering coefficient in liver and in tumor at both wavelengths (P < 0.025) and decreased the anisotropy in tumor (P < 0.025), suggesting that for modelling the dosimetry of PDT the optical properties of photosensitized tissue should be used.