Light-induced cytotoxicity after aminolevulinic acid treatment is mediated by heme and not by iron.

Photodynamic therapy (PDT) is a promising antitumor treatment strategy. However, effectiveness of PDT is limited due to an initiation of rescue responses in tumor cells, including the induction of heme oxygenase-1 (HO-1). Furthermore, the main sources of free radical production in PDT-induced oxidative stress are not clear. Here, human melanoma cells were loaded with the photosensitizer 5-aminolevulinic acid and exposed to non-thermal light of 420-800 nm at different doses. It was shown that inhibition of HO-1 activity by zinc protoporphyrin IX increased PDT-induced cytotoxicity in a dose-dependent manner. Interestingly, the cytotoxic effects were not diminished by the simultaneous application of the iron chelator desferrioxamine. Importantly, PDT together with non-toxic doses of hemin increased the number of dead cells. From these results can be concluded that heme but not iron act as the main source of free radicals in PDT treatment. This is supported by the fact that during PDT ferritin is readily up-regulated, able to bind excess iron formed by the HO-1 action. The combined treatment of photosensitizers with HO-1 inhibitors might increase the effectiveness of PDT in tumor treatment.

Inhibition of heme oxygenase-1 increases responsiveness of melanoma cells to ALA-based photodynamic therapy.

Based on the observation that 5-aminolevulinic acid (ALA) induces the expression of heme oxygenase-1 (HO-1) in cultured melanoma cells, the role of HO-1 on the effectiveness of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) was examined. Transcriptional activation of the HO-1 gene is considered to be an adaptive response to oxidative and cellular stress and confers a protective capacity against cell and tissue injury, which could affect the responsiveness to ALA-PDT. A time-dependent accumulation (0-16 h) of protoporphyrin IX (PPIX) within melanoma cells was seen after incubation with ALA (0.5 mM ALA). Over the same time interval, a significant increase (up to 25-fold) in HO-1 protein expression was observed. Thus, the production and degradation of PPIX (via heme by HO-1) were simultaneously enhanced, leading to a reduced intracellular concentration of the photodynamically active substance PPIX. Diminishing HO-1 activity by the HO-1 inhibitor tin protoporphyrin IX (SnPPIX) significantly enhanced the formation of PPIX up to 1.8 fold. A further strong increase in HO-1 protein expression (up to 128-fold) was seen after ALA-PDT treatment. Induction of HO-1 is an essential step in the 'rescue response' of tumor cells. The pharmacological inhibition of HO-1 activity by SnPPIX leads to a considerable increase in the sensitivity of tumor cells to ALA-PDT treatment. At low radiation doses (0.42 J/cm(2)), the percentages of death cells increased significantly from 7.3+/-1.3% to 43.7+/-6.4%. This effect could be further intensified by cellular depletion of HO-1 mRNA by siRNA. The combination of pharmacological inactivation of HO-1 with gene silencing led to an increase in the death rate of up to 54.1+/-8.6%. The results presented indicate that HO-1 can play a protective role against ALA-PDT mediated cytotoxicity so that a specific inhibition of HO-1 activity and/or expression might be used to increase the efficacy of ALA-based photodynamic therapy.

No evidence for prooxidative effects of homocysteine in vascular endothelial cells.

Many epidemiological studies predict a role for homocysteine (HCys) in cardiovascular disease occurrence, progression, and risk factors. In vitro studies demonstrated that HCys is an atherogenic determinant that promotes oxidant stress, inflammation, endothelial dysfunction and cell proliferation. This study originally attempted to examine the mechanism by which exposure of endothelial cells to HCys (0-250 microM) initiates inflammatory reaction and oxidative stress, by (i) investigating whether physiological and pathophysiological concentrations of HCys exhibit a prooxidative activity in vitro, (ii) examining the interaction of monocyte adhesion (Mono Mac 6) to monolayers of human microvascular endothelial cells (HMEC-1) exposed to different HCys concentrations, and (iii) examining if adherent monocytes increase reactive oxygen species either in endothelial cells or in monocytes themselves. However, our results demonstrate that HCys had neither prooxidative nor cytotoxic effects on endothelial cells. Only a moderate time- and concentration-dependent increase in monocyte adhesion up to 28.3 +/- 5.5% was achieved relative to control after 4 h of HCys stimulation. This effect was accompanied by an increased VCAM and ICAM-1 mRNA expression. This "proinflammatory" effect appeared also when HMEC-1 cells were incubated with cysteine or glutathione at the concentration range 0-250 microM, demonstrating a non-specific rather than a specific HCys effect. In addition, adherent monocytes did not increase ROS formation neither in endothelial cells nor in monocytes themselves, indicating no direct or indirect cytotoxic or prooxidative effects of HCys.

Protective effects of tomato extract with elevated beta-carotene levels on oxidative stress in ARPE-19 cells.

Epidemiological studies show that dietary products rich in carotenoids delay the progression of age-related macular degeneration. Experimental evidence from cellular studies on the antioxidant actions of carotenoids in the retinal pigment epithelium is still, however, fragmentary. The present study examined the uptake and protective potential of dietary carotenoids from tomato on the human retinal pigment epithelial cell line ARPE-19. ARPE-19 cells were incubated in medium supplemented with tomato extract containing high levels of beta-carotene, lycopene and traces of lutein. The cellular uptake of carotenoids was analysed by reverse-phase HPLC. Oxidative stress was induced by treatment with 1 mm-H2O2. Nitrotyrosine was detected by immunocytochemistry, and oxidised proteins (protein carbonyls) were measured by a quantitative ELISA method. Lipid peroxidation was assessed by quantifying thiobarbituric acid reactive substances. ARPE-19 cells preferentially accumulated lutein and beta-carotene rather than lycopene. Nitrotyrosine formation was considerably reduced in cells incubated with tomato extract compared with controls after H2O2 treatment. Protein carbonyls were reduced by 30 % (P = 0.015), and the formation of thiobarbituric acid-reactive substances was reduced by 140 % (P = 0.003) in cells incubated with tomato extract. The present study provides the experimental evidence for protective effects of dietary tomatoes rich in carotenoids on oxidative stress in the retinal pigment epithelium.

Ascorbic acid suppresses cell death in rat DS-sarcoma cancer cells induced by 5-aminolevulinic acid-based photodynamic therapy.

Especially in the public, vitamin C is considered supportive for the treatment of cancer and supplementation is common. However, the underlying mechanism that most chemotherapeutic agents, ionizing radiation, and photodynamic therapy exert on tumor cell kill is an increased production of reactive oxygen species (ROS) leading to irreversible tissue injury. Therefore, antioxidants like ascorbic acid (AA) may prevent cancer cells of cellular free radical damage and may therefore be contraindicated in patients undergoing tumor treatment. We report on the effects of AA on markers of oxidative stress and apoptosis in rat DS-sarcoma cells on 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). AA dose-dependently protected cancer cells against lipid and protein oxidation caused by ALA-PDT treatment. By real-time RT-PCR analysis an impressive increase of FasL (124-fold) and TNF-alpha (121-fold) mRNA was detected after PDT treatment. In addition, a decrease in mitochondrial transmembrane potential followed by the mitochondrial release of apoptosis-inducing factor (AIF) was observed. All these early signs of apoptosis were significantly reduced by AA, resulting in a 2.1-fold increased cell survival rate on ALA-PDT treatment. In conclusion, AA functions as a potent antioxidant, protecting mitochondria and other cell structures of oxidative cell injury induced by ALA-PDT and may therefore be contraindicated in patients undergoing tumor treatment.