Effect of photodynamic therapy mediated by hematoporphyrin derivatives on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells.

To investigate the effects of photodynamic therapy (PDT) mediated by hematoporphyrin derivatives (HPD) on the proliferation of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells. H446 cells and BEAS-2B cells were cultured in vitro with different concentrations of HPD(0, 5, 10, 12, 15, 20 µg/mL) for 4 h, and then irradiated with 630 nm laser with different energy densities (0, 25, 50, 75, 100 mW/cm2). Cell viability of H446 cells and BEAS-2B cells were detected by CCK8 assay. The cell apoptosis was observed with Annexin V-FTTC/PI double staining and Hoechst 33258. The RT-PCR examination was applied to detect the transcriptional changes of the mRNA of Bax、Bcl-2, and Caspase-9. The results of CCK8 showed that when the HPD was 15 µg/mL and the laser power density reached 50 mW/cm2, the cell viability was significantly decreased compared with the black control group. Hoechst 33258 staining showed that with the increase of HPD concentration, the cell density was reduced, and apoptotic cells increased. Flow cytometry assay revealed that the apoptotic rates of the HPD-PDT group of H446 cells and BEAS-2B cells were significantly different from those of the blank control group. The RT-PCR examination showed that the expression levels of Bax and Caspase-9 mRNA in the HPD-PDT group were up-regulated, while the expression levels of Bcl-2 mRNA were down-regulated significantly. HPD-PDT can inhibit H446 cells and BEAS-2B cells growth. The mechanism may be related to up-regulating the expression levels of Bax and Caspase-9 mRNA and down-regulating the expression levels of Bcl-2 mRNA.

Light dose effect of photodynamic therapy on growth inhibition and apoptosis induction in non-small cell lung cancer: A study in nude mouse model.

BACKGROUND: Photodynamic therapy (PDT) is receiving increasing attention in treating non-small cell lung cancer (NSCLC) worldwide, but in clinical practice, the relationship between treatment effect and PDT light dose in NSCLC remains unclear. Therefore, we aimed to determine the optimal light dose for PDT by exploring molecular biomarkers and evaluating tumor growth data. METHODS: We applied bioinformatics to identify promising genes and pathways in NSCLC and PDT. Then, the human lung adenocarcinoma cell line A549-bearing BALB/c nude mice were treated with hematoporphyrin derivative (HPD, 3 mg/kg) that is currently used widely for lung cancer treatment in the world even with photosensitization issues. After 48 h, tumor-bearing mice were irradiated superficially at doses of 100, 200, 300, 400, and 500 J/cm2. The tumor growth data and apoptotic molecules were assessed and calculated. RESULTS: Bioinformatics results indicated that the apoptosis pathway was significantly enriched and caspase 3 was the most promising biomarker on prognosis in NSCLC-PDT. Compared to the untreated group, there was no difference in the relative tumor volume (RTV) of the 100 J/cm2 group, while the RTV of the other treatment groups (200-500 J/cm2) was significantly lower. In the 100 J/cm2 group, there were significant differences in the complete remission (CR, 0 %) and the percentage of tumor growth inhibition rate (TGI%) over 75 % (20 %) compared with the other treatment groups, especially the 300 and 400 J/cm2 groups (CR 70 %; TGI% 90 %). In the 300 and 400 J/cm2 groups, the expression of caspase 3, cleaved-caspase 3, PARP1, and Bax was increased significantly, while Bcl-2 expression was significantly lower. CONCLUSIONS: Moderate doses of PDT (300 or 400 J/cm2) are more effective than low (100 or 200 J/cm2) or high doses (500 J/cm2) in the A549 tumor-bearing mice model. Since the A549 tumor is more akin to human tumors in pathological behavior, these experimental data may contribute to improving HPD-PDT illumination protocols for favorable clinical outcomes.

The study of killing effect and inducing apoptosis of 630-nm laser on lung adenocarcinoma A549 cells mediated by hematoporphyrin derivatives in vitro.

To investigate the killing effect and inducing apoptosis of 630-nm laser mediated by hematoporphyrin derivatives (HPD) on human lung adenocarcinoma A549 cells. The human lung adenocarcinoma A549 cells were incubated at random with different concentrations of HPD (5, 10, 12, 15, 20 µg/ml) for 4 h and then illuminated by 630-nm laser with different energy densities (25, 50, 75, 100 mW/cm2). And, meanwhile, the simple photosensitizer group, laser irradiation group, and blank control group were established. Then, CCK8, Hoechst 33258 staining, RT-PCR, and Western blot were employed. HPD-PDT proved no killing effect on the lung adenocarcinoma A549 cells with photosensitizer or laser irradiation alone. With the combination, the killing effect was obvious. CCK8 showed that the A549 cell viability in 15 µg/ml and 20 µg/ml HPD group as well as 50 mW/cm2, 75 mW/cm2, and 100 mW/cm2 power density group decreased significantly compared with the control group. Hoechst 33258 staining showed that with the increase of HPD concentration, the cells presented chromatin fixation and hyperchromatic nuclei. The Annexin V-FITC/PI double staining was used to detect the apoptosis rate, and the difference was statistically significant. RT-PCR and Western blot showed that the expression of Caspase-3 and Bax were significantly up-regulated. However, the Bcl-2 and Survivin were significantly down-regulated in the HPD-PDT group, while those of the other three groups showed no significant changes. HPD-PDT has a significant effect on A549 cells. The mechanism of action may be related to the upregulation of the expression of Caspase-3, Bax, and downregulation of the expression of Bcl-2 and Survivin.

The study of effect and mechanism of 630-nm laser on human lung adenocarcinoma cell xenograft model in nude mice mediated by hematoporphyrin derivatives.

To investigate the effect and mechanism of 630-nm laser on human lung adenocarcinoma cell xenograft model in nude mice mediated by hematoporphyrin derivatives (HPD) and provide theoretical basis for clinical photodynamic therapy (PDT). Human lung adenocarcinoma cell xenograft model in nude mice was established and randomly divided into four groups: control group, pure photosensitizer group, pure irradiation group, and photodynamic treatment group. The tumor volume growth was compared, and the tumor growth inhibition rate was calculated. HE staining was used for routine pathological observation of tumor sections, and gross conditions of cells, interstitium, and blood vessels in several groups of tumor tissues were observed. TUNEL staining was used to observe and compare the apoptosis induced by photodynamic therapy. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression level of angiogenesis-related factors VEGF, HIF-1α and apoptosis-related factors Bax and Bcl-2 mRNA in the transplanted tumor tissues. Western blot was employed to detect the expression of angiogenesis-related proteins VEGF, HIF-1α and apoptosis-related proteins Bax, Caspase-3, and Bcl-2. Compared with the other three groups, the tumor growth inhibition rate of the photodynamic treatment group was significantly increased and the difference was statistically significant (P < 0.05). HE staining showed that the animal model of lung adenocarcinoma A549 was successfully established. TUNEL staining revealed that more apoptotic cells were found in the photodynamic treatment group, and the apoptosis index was calculated. Compared with the other three groups, the difference was statistically significant (P < 0.05). RT-PCR results showed that compared with the other three groups, the mRNA expressions of VEGF, HIF-1α, and Bcl-2 in the photodynamic treatment group decreased, while the expression of Bax mRNA increased(P < 0.05), and the differences were statistically significant. Western blot results showed that protein expressions of VEGF, HIF-1α, and Bcl-2 decreased in the photodynamic treatment group, while protein expression level of Bax and Caspase-3 increased (P < 0.05), indicating statistically significant differences. The 630-nm laser mediated by hematoporphyrin derivatives can significantly inhibit the growth of human lung adenocarcinoma xenograft tumor in nude mice, the mechanism of which is related to the inhibition of tumor angiogenesis by down-regulating VEGF and HIF-1α gene expression, and the promotion of tumor apoptosis by up-regulating Bax, Caspase-3, and down-regulating Bcl-2 gene expression.

Identification of the targets of hematoporphyrin derivative in lung adenocarcinoma using integrated network analysis.

BACKGROUND: Hematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma. METHODS: RNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using "BioCloud" online tool. Protein-protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software. RESULTS: A total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated-treated with X-ray-X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429. CONCLUSION: RPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.

Identification of the targets of hematoporphyrin derivative in lung adenocarcinoma using integrated network analysis

BACKGROUND: Hematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma. METHODS: RNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using "BioCloud" online tool. Protein-protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software. RESULTS: A total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated-treated with X-ray-X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429. CONCLUSION: RPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.

Photodynamic therapy (PDT) for malignant brain tumors--where do we stand?

INTRODUCTION: What is the current status of photodynamic therapy (PDT) with regard to treating malignant brain tumors? Despite several decades of effort, PDT has yet to achieve standard of care. PURPOSE: The questions we wish to answer are: where are we clinically with PDT, why is it not standard of care, and what is being done in clinical trials to get us there. METHOD: Rather than a meta-analysis or comprehensive review, our review focuses on who the major research groups are, what their approaches to the problem are, and how their results compare to standard of care. Secondary questions include what the effective depth of light penetration is, and how deep can we expect to kill tumor cells. CURRENT RESULTS: A measurable degree of necrosis is seen to a depth of about 5mm. Cavitary PDT with hematoporphyrin derivative (HpD) results are encouraging, but need an adequate Phase III trial. Talaporfin with cavitary light application appears promising, although only a small case series has been reported. Foscan for fluorescence guided resection (FGR) plus intraoperative cavitary PDT results were improved over controls, but are poor compared to other groups. 5-Aminolevulinic acid-FGR plus postop cavitary HpD PDT show improvement over controls, but the comparison to standard of care is still poor. CONCLUSION: Continued research in PDT will determine whether the advances shown will mitigate morbidity and mortality, but certainly the potential for this modality to revolutionize the treatment of brain tumors remains. The various uses for PDT in clinical practice should be pursued.

Photodynamic efficiency of hypericin compared with chlorin and hematoporphyrin derivatives in HEp-2 and Vero epithelial cell lines.

Hypericin (HY) is a photoactive aromatic dianthraquinone that is considered a potent photodynamic agent. In this study, hypericin and two other photosensitizers, a hematoporphyrin derivative (Photogem(®); PG) and a chlorin derivative (Photodithazine(®); PZ), were compared in terms of their phototoxicity toward two cell lines, HEp-2 and Vero. The median inhibitory concentration (IC(50)) of each of the photosensitizers was obtained after a 16.2J cm(-2) dose of irradiation at 630 ± 10 nm. The IC(50) values were 0.07 ± 0.01 (HY), 1.0 ± 0.2 (PZ), and 9 ± 1 µgmL(-1) (PG) in HEp-2 cells and 0.3 ± 0.1 (HY), 1.6 ± 0.2 (PZ) and 11 ± 1 µgmL(-1) (PG) in Vero cells, showing that HY is more phototoxic than the others when irradiated at 630 nm. If these results are analyzed, simultaneously, with the first-order constant for BSA tryptophan photooxidation, obtained by fluorescence decay (λ(excitation)=280 nm), which are 11×10(-3) min(-1)±1. 10(-3) min(-1) (HY), 10 × 10(-3) min(-1)±1 × 10(-3) min(-1) (PZ), and 6 × 10(-3)min(-1) ± 1×10(-3)min(-1) (PG), it is possible to infer that the photodynamic efficiency alone is not sufficient to explain the higher HY phototoxicity. The lipophilicity is also an important factor for an efficient target cell accumulation and was assessed for all sensitizers through the octanol-water partition coefficient (log P): 1.20 ± 0.02 (HY), -0.62 ± 0.03 (PZ), and -0.9 ± 0.2 (PG). The higher value for HY correlates well with its observed superior efficiency to promote damage at low concentrations and doses. As HY is used for the long-term treatment of mild depression, it is considered safe for humans. This fact and the present results reinforce the great potential of this photosensitizer to replace porphyrin derivatives, with the advantages that mean it could be used as photosensitizer in clinical photodynamic therapy.

Imbalance of Ca2+ and K+ fluxes in C6 glioma cells after PDT measured with scanning ion-selective electrode technique.

Photodynamic therapy (PDT) possesses the capacity to lead to death of C6 glioma in vitro and in vivo. The purpose of this study was to investigate whether Ca(2+) and K(+) homeostasis of C6 glioma cells were affected by PDT. C6 glioma cells were randomly divided into five groups: control group, Hematoporphyrin derivative (HpD) group (10 mg/l, without irradiation), PDT group (HpD 10 mg/l + irradiation), PDT&6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) group (HpD 10 mg/l + CNQX 50 mol/l + irradiation), and HpD&CNQX group (HpD 10 mg/l + CNQX 50 mol/l, without irradiation). Glioma cells in PDT and PDT&CNQX group were subjected to PDT. Cells in PDT&CNQX group were administered α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist CNQX prior to PDT on C6 glioma cells. The changes of Ca(2+) and K(+) fluxes were studied by using a non-invasive scanning ion-selective electrode technique (SIET). Morphology of C6 cells was observed with optical microscopy. PDT induced Ca(2+) influx and K(+) efflux significantly, which resulted in death of C6 cells. When AMPA glutamate receptor antagonist CNQX was applied, Ca(2+) influx and K(+) efflux were partly blocked up and viability of C6 cells increased. These results indicate that Ca(2+) influx and K(+) efflux may correlate with the treatment effects of PDT on C6 glioma cells.

Photodynamic therapy for the treatment of induced mammary tumor in rats.

The objective of this work was to evaluate photodynamic therapy (PDT) by using a hematoporphyrin derivative as a photosensitizer and light-emitting diodes (LEDs) as light source in induced mammary tumors of Sprague-Dawley (SD) rats. Twenty SD rats with mammary tumors induced by DMBA were used. Animals were divided into four groups: control (G1), PDT only (G2), surgical removal of tumor (G3), and submitted to PDT immediately after surgical removal of tumor (G4). Tumors were measured over 6 weeks. Lesions and surgical were LEDs lighted up (200 J/cm(2) dose). The light distribution in vivo study used two additional animals without mammary tumors. In the control group, the average growth of tumor diameter was approximately 0.40 cm/week. While for PDT group, a growth of less than 0.15 cm/week was observed, suggesting significant delay in tumor growth. Therefore, only partial irradiation of the tumors occurred with a reduction in development, but without elimination. Animals in G4 had no tumor recurrence during the 12 weeks, after chemical induction, when compared with G3 animals that showed 60 % recurrence rate after 12 weeks of chemical induction. PDT used in the experimental model of mammary tumor as a single therapy was effective in reducing tumor development, so the surgery associated with PDT is a safe and efficient destruction of residual tumor, preventing recurrence of the tumor.

Safety assessment of oral photodynamic therapy in rats.

Photodynamic therapy (PDT) is based on the synergism of a photosensitive drug (a photosensitizer) and visible light to destroy target cells (e.g., malignant, premalignant, or bacterial cells). The aim of this study was to investigate the response of normal rat tongue mucosa to PDT following the topical application of hematoporphyrin derivative (Photogem®), Photodithazine®, methylene blue (MB), and poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with MB. One hundred and thirty three rats were randomly divided in various groups: the PDT groups were treated with the photosensitizers for 10 min followed by exposure to red light. Those in control groups received neither photosensitizer nor light, and they were subjected to light exposure alone or to photosensitizer alone. Fluorescent signals were obtained from tongue tissue immediately after the topical application of photosensitizers and 24 h following PDT. Histological changes were evaluated at baseline and at 1, 3, 7, and 15 days post-PDT treatment. Fluorescence was detected immediately after the application of the photosensitizers, but not 24 h following PDT. Histology revealed intact mucosa in all experimental groups at all evaluation time points. The results suggest that there is a therapeutic window where PDT with Photogem®, Photodithazine®, MB, and MB-loaded PLGA nanoparticles could safely target oral pathogenic bacteria without damaging normal oral tissue.

The influence of electroporation on in vitro photodynamic therapy of human breast carcinoma cells.

Phototoxicity of drugs used in cancer photodynamic therapy could be augmented by increased accumulation of a photosensitizer in target cells. The intracellular delivery mode that enhances drug transportation could facilitate therapy by reducing the exposure time. Doses of the administered drug and related side effects could be lowered, whilst maintaining the same therapeutic efficiency. Electroporation supports transport of many drugs by creating electric field-induced transient nanopores in the plasma membrane. In this study, the electroporation- assisted transport of a photosensitizer was tested in vitro in human breast carcinoma cell lines: wild-type (MCF-7/WT) and doxorubicin-resistant (MCF-7/DOX). The efficacy of photodynamic therapy alone and in combination with electroporation was evaluated by cell viability with MTT test, using a haematoporphyrin derivative as a model. The data presented show up to 10-fold greater efficacy of the combined method, with very significantly reduced drug exposure times.

Photo-oxidative action in cervix carcinoma cells induced by HPD - mediated photodynamic therapy.

UNLABELLED: Photodynamic therapy leads to oxidative stress through the generation of free radicals. Oxidative stress causes damage to cellular macromolecules such as nucleic acids, proteins and lipids. AIM: To examine the hematoporphyrin derivative (HpD) - mediated photodynamic effect on cervical adenocarcinoma cell line HeLa. METHODS: The HpD localization in HeLa cells was analyzed by confocal microscopy with epi-fluorescence system. Lipid peroxidation (LPO) was estimated by measurement of the concentration of malondialdehyde, protein degradation - by modified Ellman's method, superoxide dysmutase (SOD) - using Ransod Kit. The expression of inducible nitric oxide synthase (iNOS) was detected by immunocytochemical staining. RESULTS: The HpD was distributed all over the cytoplasm with preferential localization in the inner side of the plasma membrane and around the nuclear envelope. The process of photosensitizer distribution was time dependent. PDT-HpD increased the level of malonodialdehyde (MDA), SOD activity and the expression of iNOS in HeLa cells. However, PDT induced the decrease in the level of protein-associated thiol groups. CONCLUSIONS: Our study showed the important role of PDT-mediated oxidative stress in HeLa cells. HpD-PDT might be alternative and less invasive approach for treatment of patients with cervical cancer resistant for standard chemotherapy and radiotherapy.

[Main factors affecting the effect of photodynamic therapy against human esophageal cancer cells in vitro].

OBJECTIVE: To evaluate the tumor cell-killing effect of photodynamic therapy against human esophageal cancer cells in vitro and identify the main factors affecting the effect. METHODS: Human esophageal cancer Eca-109 cells were incubated for 24 h in vitro with hematoporphyrin derivative (HpD) and Photofrin at different concentrations prior to exposure to a light energy density of 15 J/cm(2) delivered from a DIOMED 630 PDT system. The cell killing effect was also evaluated for different HpD concentrations combined with 3 light energy densities (10, 30, and 50 J/cm(2)), respectively. The cell survival rate was measured using MTT assay, and fluorescence spectrometry was used to detect the intracellular photosensitizer fluorescence of the tumor cells after incubation with HpD for 4 h. RESULTS: The cell survival rate after incubation with the two photosensitizers at different concentrations were significantly different, and under the 3 different light energy densities, incubation of the cells with different HpD concentrations also resulted in significantly different cell survival rates (P<0.05). At the 4 low photosensitizer concentrations and with different light energy densities, the cell survival rates were similar (P>0.05), but the 4 higher photosensitizer concentrations resulted in significant difference in the cells survival (P<0.05). Correlation analysis showed that the intracellular photosensitizer concentration was positively correlated to the photosensitizer concentrations in cell incubation (r=0.997). CONCLUSION: When the light source remains constant, the light energy density, the kinds of photosensitizers and their concentrations are the main factors affecting the Eca-109 cell-killing effect of PDT.

[Hematoporphyrin derivative-mediated photodynamic therapy for human nasopharyngeal carcinoma: a comparative study with CNE2 and C666-1 cell lines in vitro].

OBJECTIVE: To investigate biological effect of hematoporphyrin derivative (HpD) photodynamic therapy (PDT) on in vitro cultured nasopharyngeal carcinoma (NPC) cell lines CNE2 and C666-1. METHODS: CNE2 and C666-1 cells cultured in vitro were incubated in a medium containing HpD at different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 microg/ml) for 4 h followed by exposure to different light doses (2, 5, 10, and 20 J/cm2) using a diode laser at 630 nm with power density of 20 mW/cm2. After 24 h of incubation with HpD-PDT, the survival rate of CNE2 and C666-1 cells were analyzed by MTT assay. RESULTS: HpD-PDT produced effective killing of CNE2 and C666-1 cells cultured in vitro, and the killing effects were positively correlated with HpD concentration and the irradiation dose. Exposure of CNE2 and C666-1 cells to irradiation dose of 20 J/cm2 resulted in the IC50 of 0.7 and 1.2 microg/ml, respectively (P<0.01). With the same HpD concentration and irradiation dose, the survival rate of C666-1 cells, however, was significantly higher than that of CNE2 cells (P<0.05). CONCLUSION: HpD-PDT may result in effective killing of CNE2 and C666-1 cells cultured in vitro, although C666-1 cells are less sensitive to HpD-PDT than CNE2 cells.

Hydrogen peroxide, superoxide, and hydroxyl radicals are involved in the phototoxic action of hematoporphyrin derivative against tumor cells.

This study was aimed to estimate the participation of reactive oxygen species (ROS), other than singlet oxygen (1O2), in the antitumor effect of photodynamic therapy (PDT) with hematoporphyrin derivative (HPD) as well as to determine the ability of photoexcited HPD to the formation of protein peroxides that currently are regarded as a new form of ROS. Studies were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in phosphate-buffered saline and then irradiated with red light at 630 run in the same buffer. Experiments indicated that H2O2 and oxygen radicals could mediate the tumoricidal action of HPD-PDT; we found that photosensitization of EAC cells with HPD leads to the formation of significant amounts of H2O2, superoxide (O2-.), and hydroxyl (OH.) radicals, which along with 1O2 were involved in photoinactivation of the cells in vitro. Our data showed that in EAC cells subjected to HPD-PDT, the generation H2O2, O2-., and OH. could be largely mediated by: (i) an increase in the activity of xanthine oxidase (XOD), due most probably to the conversion of xanthine dehydrogenase (XDH) to XOD via a Ca2+-dependent proteolytic process as well as oxidation of SH groups in XDH; and (ii) photooxidation of some cellular constituents (proteins). Another interesting finding of our studies is that in tumor cells subjected to HPD-PDT the Fenton-like reactions could play an important role in the generation of OH., and that cell-bound Cu/Zn-superoxide dismutase as well as catalase can protect tumor cells against the phototoxic action of HPD. In addition, we clearly demonstrated the ability of photoexcited HPD to the generation of protein peroxides in tumor cells. Studies suggest that 1O2 is the main agent responsible for the generation of protein peroxides in EAC cells treated with HPD-PDT, although other ROS (H2O2, O2-., and OH.) were also implicated in this process. However, further work is needed to clarify the significance of these peroxides in the antitumor effect of PDT with HPD.

Study of solar photosensitization processes on dermatophytic fungi.

The antifungal activity of solar simulator was evaluated in presence of haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) as photosensitizers. Seven dermatophytes were used as test fungi. The solar simulator at fluence rate 400 W/m2 for 30 minutes induced marked inhibition for spore germination of the photosensitized fungi. The rate of inhibition varied according to the fungal species and concentration of the photosensitizer. There was an increase in percentage inhibition of spore germination as the concentration of HPD or MB increased. Complete inhibition for spore germination of Trichophyton. verrucosum, T. mentagrophytes, and Miccrosporum canis was induced when these species were pretreated with 10(-3) M of HPD or MB before irradiation. Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. On contrary, the maximum reduction in percentage spore germination was induced at the lowest concentration (10(-7) M) of TBO. The tested dermatophytes were mostly capable of producing different enzymes (keratinase, phosphatases, amylase, lipase). The separate application of radiation or photosensitizer was ineffective or exerted slight inhibition on enzyme production. However, the activity of the enzymes was drastically inhibited when the fungi were irradiated after their treatment with photosensitizer. T. verrucosum and T. mentagrophytes were the most sensitive. In a trail to apply a control measure against dermatomycosis using solar simulator radiation, the results revealed that the radiation was successful in curing the MB-photosensitized guinea pigs, artificially infected with T. verrucosum, T. mentagrophytes or M. canis. The percentage of recovery reached 100% in some treatments.

Effects of photodynamic treatment on biological antioxidant systems in rats.

Effects of photodynamic treatments on inherent antioxidant metabolites and cellular defence enzymes have been investigated in rats. Wistar rats were grouped into untreated controls, light controls, hematoporphyrin derivative (Hpd) (treated with 5 and 10 mg Hpd/kg body weight and kept in dark) and sets treated with both Hpd and red light (dose 172 and 344 j/m2 ). After 2, 24, 48 and 72 hr of Hpd injection the rats sacrificed, livers quickly excised to analyze Hpd uptake, activities of enzymes like catalase, GSH-Px and antioxidants like GSH, vitamin A, vitamin E and vitamin C. The results showed that the loss of Hpd from liver as a function of post- injection time was non- linear. An increased generation of lipid radicals was observed in the groups treated with 5 mg Hpd and higher dose of light and in groups treated with 10 mg Hpd at both the doses of light. Combination of light and Hpd reduced hepatic GSH content with a concomitant reduction in GSH-Px. At higher doses of Hpd and light, there was a significant reduction in hepatic vitamin A levels. Combination of Hpd and light in all doses reduced vitamin E content in liver. The decreased biological antioxidant contents and GSH-Px may be attributed to their utilization for the scavenging of free radicals generated by Hpd and light in tissues. However, no change in catalase activity and vitamin C content in liver was noted in experimental rats. The results suggest that exposure to higher doses of Hpd with light alters oxidant stress system and TBARS content in rat.

Determination of endogenous porphyrins and the maximal HpD tumor/normal skin ratio in SKH-1 hairless mice by light induced fluorescence spectroscopy.

The treatment of skin tumors is an application of photochemotherapy (PCT) which involves an initial administration of a photosensitizer (PS) followed by irradiation with a light beam that causes the PS to produce cytotoxic oxygen species within the tumors. As the PS is also present in normal skin, it is necessary to know how it is distributed between the two tissues. In this study, we have used SKH-1 hairless mice bearing papillomas or carcinomas chemically induced. The biodistribution of hematoporphyrin derivative (HpD) and the tissue autofluorescence measurements were studied by light induced fluorescence spectroscopy. The tumor and normal autofluorescence spectra measured on control mice with papillomas or carcinomas had a very similar shape. However, the principal endogenous porphyrin peak at about 630 nm showed a fluorescence signal amplitude 2 (for papilloma) and 1.5 (for carcinoma)-fold higher than the one found for the normal skin. Moreover, the fluorescence intensity of carcinoma spectrum is 1.4-fold lower than the one of papilloma spectrum at 630 nm. The tissue autofluorescence can be used to distinguish tumor from normal skin and benign from malignant tumor. This difference in fluorescence intensity at 630 nm was directly related to the concentration of endogenous porphyrins in the tumor. Fluorescence intensity ratios between tumor and normal skin were measured 4, 8, 24, 48, 72 and 96 hours after intraperitoneal injection of HpD (5 mg/kg body weight). The best tumor/normal skin ratio was 6.2 for HpD and the time required to reach this ratio was 48 h. HpD showed a moderate selectivity since the ratio was higher than 1 during the four first days. Photodynamic therapy with the same dose of HpD used in this biodistribution study must also be carried out to verify that the maximal tumor/skin ratio corresponds to the maximal efficiency of HpD.