The Role of Lipid Peroxidation and Protein Degradation in the Photodestruction of Ehrlich Ascites Carcinoma Cells Sensitized by Hematoporphyrin Derivative (original) (raw)
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Journal of Photochemistry and Photobiology B: Biology, 2008
Hematoporphyrin derivative (HPD), a sensitizer used in photodynamic therapy (PDT) of malignancies, is progressively destroyed during the treatment. Prior studies suggested that upon PDT the photobleaching of HPD in tumor tissues is largely mediated by self-sensitized singlet oxygen. However, little is known about the role of other reactive oxygen species (ROS). The main aim of this work was to clarify the significance of H 2 O 2 , superoxide (O ÅÀ 2) and hydroxyl (OH Å) radicals in bleaching of HPD in tumor cells subjected to PDT. Experiments were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in PBS and then irradiated with red light at 630 nm in the same buffer. Studies showed that photosensitization of EAC cells by HPD led to the formation of significant amounts of H 2 O 2 , O ÅÀ 2 and OH Å , and that these ROS could be involved in the photobleaching of HPD during PDT. In fact, we found that addition of catalase (CAT, a scavenger H 2 O 2), Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Tiron (scavengers of O ÅÀ 2), Na-benzoate, mannitol and deferoxamine (scavengers of OH Å) caused a substantial decrease in the rate of HPD photobleaching in EAC cells. In these cells, the inhibitory effects of Na-benzoate, mannitol and deferoxamine on the photodegradation of HPD correlated well with suppression of the OH Å generation, a highly active oxidizer. In EAC cells, the glutathione redox cycle and CAT (scavengers of H 2 O 2) as well as Cu/Zn-SOD was found to suppress the photoinduced degradation of HPD. It was also established that HPD can directly scavenge H 2 O 2 and oxygen free radicals; in a phosphate buffer its second-order rate constants were measured as 5.51 ± 0.32 Â 10 3 M À1 s À1 (for the reaction with O ÅÀ 2), 5.08 ± 0.31 Â 10 4 M À1 s À1 (for H 2 O 2), and 3.44 ± 0.08 Â 10 10 M À1 s À1 (for OH Å). Thus, our data suggest that OH Å could be one of the main oxidants mediating the photobleaching behavior of HPD in malignancies. Studies showed that photoexcited moieties of HPD can oxidize cell proteins with the formation of protein peroxides (PPO), which currently are regarded as a new form of ROS. Model experiments suggest that PPO could also participate in bleaching of HPD in tumors treated with PDT. It was found that HPD may destroy in tumor cells after cessation of photoirradiation and that this event is largely mediated by the presence of H 2 O 2 , a precursor of OH Å .
Journal of Environmental Pathology Toxicology and Oncology, 2006
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.
Neoplasma, 2007
The aim of this study was to clarify the mechanism of the stimulatory effect of heat stress on generation of superoxide radical (O2-*) in tumors subjected to photodynamic therapy (PDT) with hematoporphyrin derivative (HPD). For this purpose, the effect of heating on the activity of xanthine oxidase (XOD) in tumor cells upon their photosensitization with HPD was examined; this enzyme is participated in purine catabolism and has the ability to generate O2-*, a precursor of H2O2 and very cytotoxic hydroxyl radical. The study was carried out on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in a serum-free medium and then irradiated with red light (lambda max = 630 nm) at 3 different temperatures (30, 37 and 44 degrees C). In the cells, the activity of XOD was assayed fluorometrically, using pterine as the substrate, whereas the production of O2-* by the nitro blue tetrazolium method. It was found that increasing of the temperature from 30 to 44 degrees C strongly (by...
Folia biologica
The oxidative effects of photodynamic therapy with 5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin (TMP) and Zn-5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin (ZnTMP) were evaluated in Wistar rats subcutaneously inoculated with Walker 256 carcinoma. The animals were irradiated with red light (λ = 685 nm; D = 50 J/cm2; 15 min) 3 h after intra-peritoneal administration of 10 mg/kg body weight of porphyrins. The presence of free radicals in tumours after photodynamic therapy with TMP and ZnTMP revealed by chemiluminescence of luminol attained the highest level at 18 h after irradiation. Lipid peroxides measured as thiobarbituric-reactive substances and protein carbonyls, which are indices of oxidative effects produced on susceptible biomolecules, were significantly increased in tumour tissues of animals 24 h after photodynamic therapy. The levels of thiol groups and total antioxidant capacity in the tumours were decreased. The activities of antioxidant enzymes superoxide dismutase and g...
Experimental oncology, 2004
To elucidate the mechanism of the potentiating influence of heating associated with photoirradiation on the antitumor efficiency of photodynamic therapy (PDT) with hematoporphyrin derivative (HPD). The study was carried out on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in a serum-free medium and then irradiated with red light (lambda max=630 nm) at various temperatures. Cytotoxicity was estimated by the trypan blue exclusion assay. Our data support the view that in PDT the hyperthermia (around 44 degrees C) produced by irradiation can enhance synergistically the HPD-photoinduced tumor eradication; it was found that raising the irradiation temperature from 30 to 44 degrees C caused a substantial (approximately 1.5 fold) increase in the rate of HPD-photosensitized inactivation of EAC cells, while hyperthermia (44 degrees C) itself showed little toxic effects towards the cells. Studies indicated that the potentiating effect of heating on the antitumor efficiency ...
Oxygen Dependence of Hematoporphyrin Derivative-induced Photoinactivation of Chinese Hamster Cells
Cancer Research, 1985
The oxygen dependence of hematoporphyrin derivative (HPD)induced photoinactivation of Chinese hamster V79 cells was examined. Cells were treated with HPD (25 ng/mï)for 2 h and subsequently exposed to red light (>590 nm) under either aer ated or hypoxic (<10 ppm 02) conditions. Hypoxie cells were found to be extremely resistant to the lethal effects of HPD and light. The electron-affinic X-ray hypoxic cell sensitizer, SR-2508, did not sensitize hypoxic HPD-treated cells to light. The clinical implications of these findings are discussed, with consideration of the possibility that hypoxic areas in tumors may limit HPD phototherapy.
Photo-oxidative action in cervix carcinoma cells induced by HPD - mediated photodynamic therapy
PubMed, 2009
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.
Energetics and efficiency of photoinactivation of murine tumor cells containing hematoporphyrin
Cancer Research, 1976
Hematoporphyrin derivative at an intracellular concentration in TA-3 mouse mammary carcinoma cells of 0.6 or 0.9 mM required input of 3.0 to 3.6 • 10" quanta/cell of red light (620 nm)to achieve a 90% kill. At an intracellular concentration of 1.2 mM, this light requirement drops to 1.5 • 10" quanta/cell. The energy for this photodynamic process is about 100 times higher than that required for ionizing radiation to achieve the same level of kill for these cells. The quantum yield for singlet oxygen formation (the cytotoxic agent in most photodynamic processes) from hematoporphyrin derivative is 0.75 -0.07 in ethanol but only 0.16 -+ 0.07 within TA-3 cells, indicating possible intracellular complexing and .quenching.
Medical science monitor : international medical journal of experimental and clinical research, 2005
The use of photodynamic therapy (PDT) to treat malignant tumors involves a photosensitizing drug, light, and oxygen. Since PDT requires oxygen, the purpose of this study was to investigate whether increased oxygen tension increases cell death compared with normoxic conditions (21 kPa oxygen). The effect of hyperoxia on PDT and lipid peroxidation was investigated in two human colon carcinoma cell lines, SW480 and WiDr, and one rat bladder cell carcinoma, AY-27. The cells were incubated with 2 mM 5-aminolaevulinic acid (5-ALA) for 3.5 h at 37 degrees C. This treatment induces the accumulation of protoporphyrin IX (PpIX), which is a potent photosensitizer. PpIX absorbs light energy and produces singlet oxygen in the presence of molecular oxygen, which then destroys the cancer cells. The cells were illuminated (0.5-30 min) at room temperature by blue light (435 nm) prior to hyperoxia exposure. This treatment was performed in a small, temperature-controlled (37 degrees C) hyperbaric cham...