Odrun A. Gederaas

5‐Aminolevulinic Acid, but not 5‐Aminolevulinic Acid Esters, is Transported into Adenocarcinoma Cells by System BETA Transporters

Abstract 5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis–Menten kinetics (Km = 8–10 mM and Vmax = 18–20 nmol·(mg protein × h)−1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32°C. The activation energy was 112 kJ·mol−1 in the temperature range 15°–32°C and 26 kJ·mol−1 above 32°C. Transport of 5-ALA was Na+ and partly Cl−-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65–75%, by the β-amino acids, β-alanine and taurine and by γ-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-β-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM β-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 μM) was not significantly influenced by β-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by β-amino acid and GABA carriers in this cell line.

Closely related colon cancer cell lines display different sensitivity to polyunsaturated fatty acids, accumulate different lipid classes and downregulate sterol regulatory element‐binding protein 1

N‐6 polyunsaturated fatty acids (PUFAs) may be associated with increased risk of colon cancer, whereas n‐3 PUFAs may have a protective effect. We examined the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid and arachidonic acid on the colon carcinoma cell lines SW480 derived from a primary tumour, and SW620 derived from a metastasis of the same tumour. DHA had the strongest growth‐inhibitory effect on both cell lines. SW620 was relatively more growth‐inhibited than SW480, but SW620 also had the highest growth rate in the absence of PUFAs. Flow cytometry revealed an increase in the fraction of cells in the G2/M phase of the cell cycle, particularly for SW620 cells. Growth inhibition was apparently not caused by increased lipid peroxidation, reduced glutathione or low activity of glutathione peroxidase. Transmission electron microscopy revealed formation of cytoplasmic lipid droplets after DHA treatment. In SW620 cells an eightfold increase in total cholesteryl esters and a 190‐fold increase in DHA‐containing cholesteryl esters were observed after DHA treatment. In contrast, SW480 cells accumulated DHA‐enriched triglycerides. Arachidonic acid accumulated in a similar manner, whereas the nontoxic oleic acid was mainly incorporated in triglycerides in both cell lines. Interestingly, nuclear sterol regulatory element‐binding protein 1 (nSREBP1), recently associated with cell growth regulation, was downregulated after DHA treatment in both cell lines. Our results demonstrate cell‐specific mechanisms for the processing and storage of cytotoxic PUFAs in closely related cell lines, and suggest downregulation of nSREBP1 as a possible contributor to the growth inhibitory effect of DHA.

Monitoring of hexyl 5-aminolevulinate-induced photodynamic therapy in rat bladder cancer by optical spectroscopy

Monitoring of the tissue response to photodynamic therapy (PDT) can provide important information to help optimize treatment variables such as drug and light dose, and possibly predict treatment outcome. A urinary bladder cancer cell line (AY-27) was used to induce orthotopic transitional cell carcinomas (TCC) in female Fischer rats, and hexyl 5-aminolevulinate (HAL, 8 mM, 1 h)-induced PDT was performed on day 14 after instillation of the cancer cells (20 J/cm(2) fluence at 635 nm). In vivo optical reflectance and fluorescence spectra were recorded from bladders before and after laser treatment with a fiberoptic probe. Calculated fluorescence bleaching and oxygen saturation in the bladder wall were examined and correlated to histology results. Reflectance spectra were analyzed using a three-layer optical photon transport model. Animals with TCC treated with PDT showed a clear treatment response; decreased tissue oxygenation and protoporphyrin IX (PpIX) fluorescence photobleaching were observed. Histology demonstrated that 3 of 6 animals with treatment had no sign of the tumor 7 days after PDT treatment. The other 3 animals had significantly reduced the tumor size. The most treatment-responsive animals had the highest PpIX fluorescence prior to light irradiation. Thus, optical spectroscopy can provide useful information for PDT. The model has proved to be very suitable for bladder cancer studies.

A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells

Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were treated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separated and quantified by high performance liquid chromatography (HPLC), both as free porphyrin acids after an easy extraction method with a subsequent reverse phase technique, and then as porphyrin esters after a more laborious extraction method and subsequent normal phase technique. The porphyrins were detected by means of a fluorescence detector. Analysis by normal phase HPLC indicated that 81% (739 pmol/mg protein) of the total amounts of fluorescing porphyrins accumulated was protoporphyrin IX, while similar analysis by reverse phase HPLC indicated that PpIX constituted 91% (622 pmol/mg protein) of the accumulated porphyrins. In addition to protoporhyrin IX, copro-, hexa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated cells by both methods. The discrepancy between the two methods increased with increasing hydrophilicity of the analysed porphyrins, with uroporphyrin estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than by reverse phase HPLC.

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.

5-Aminolevulinic acid induced lipid peroxidation after light exposure on human colon carcinoma cells and effects of α-tocopherol treatment

Abstract This work relates to studies on modes of phototoxicity by protoporphyrin (PpIX) after incubation of 5-aminolevulinic acid (5-ALA) on cultured cells. Lipid peroxidation in the 5-ALA incubated primary adenocarcinoma cells from the rectosigmoid colon (WiDr cells) was determined by measurement of protein-associated thiobarbituric acid reactive substances (TBARS). TBARS were increased 2-fold in cells treated with 2 mM 5-ALA for 3.5 h in serum enriched medium. After illumination of 5-ALA incubated cells, TBARS were formed in a light dose dependent manner. TBARS analysis were compared with high-performance liquid chromatography (HPLC) analysis of malondialdehyde, and results indicate that 90% of the thiobarbituric reactive substances were due to malondialdehyde. Pretreating WiDr cells with α-tocopherol for 48 h inhibits the cytotoxic effect of 5-ALA and increases 5-fold the light dose needed to kill 50% of the cells. Pretreatment with α-tocopherol shows a considerable decrease (about 80%) on TBARS formation after illumination. The cellular content of α-tocopherol was determined by HPLC and found to be 15.3 pmol/10 6 cells.

Gold Tris(carboxyphenyl)corroles as Multifunctional Materials: Room Temperature Near-IR Phosphorescence and Applications to Photodynamic Therapy and Dye-Sensitized Solar Cells

Two amphiphilic corroles-5,10,15-tris(3-carboxyphenyl)corrole (H3[mTCPC]) and 5,10,15-tris(4-carboxyphenyl)corrole (H3[pTCPC])-and their gold complexes have been synthesized, and their photophysical properties and photovoltaic behavior have been investigated. Like other nonpolar gold corroles, Au[mTCPC] and Au[pTCPC] were both found to exhibit room temperature phosphorescence in deoxygenated solutions with quantum yields of ∼0.3% and triplet lifetimes of ∼75 μs. Both compounds exhibited significant activity as dyes in photodynamic therapy experiments and in dye-sensitized solar cells. Upon irradiation at 435 nm, both Au corroles exhibited significant phototoxicity against AY27 rat bladder cancer cells while the free-base corroles proved inactive. Dye-sensitized solar cells constructed using the free bases H3[mTCPC] and H3[pTCPC] exhibited low efficiencies (≪1%), well under that obtained with 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin, H2[pTCPP] (1.9%, cf. N719 9.5%). Likewise, Au[pTCPC] proved inefficient, with an efficiency of ∼0.2%. By contrast, Au[mTCPC] proved remarkably effective, exhibiting an open-circuit voltage (Voc) of 0.56 V, a short-circuit current of 8.7 mA cm(-2), a fill factor of 0.72, and an efficiency of 3.5%.

Can dogs smell lung cancer? First study using exhaled breath and urine screening in unselected patients with suspected lung cancer.

Abstract Background. On the basis of our own experience and literature search, we hypothesised that a canine olfactory test may be useful for detecting lung cancer in an unselected population of patients suspected to have lung cancer. Material and methods. We conducted a prospective study of 93 patients consecutively admitted to hospital with suspected lung cancer. Exhaled breath and urine were sampled before the patients underwent bronchoscopy. The canine olfactory test was performed in a double-blinded manner. Sensitivity and specificity were outcome measures. Results. With 99% sensitivity, the olfactory test demonstrated that dogs have the ability to distinguish cancer patients from healthy individuals. With an intensified training procedure, the exhaled breath and urine tests showed sensitivity rates of 56–76% and specificity rates of 8.3–33.3%, respectively, in our heterogeneous study population. Conclusion. Although the olfactory test appears to be a promising tool for the detection of cancer, the main challenge is to determine whether the test can sufficiently discriminate between patients at risk, patients with benign disease, and patients with malignant disease. We need to gain a deeper understanding of this test and further refine it before applying it as a screening tool for lung cancer in clinical settings.

Photodynamic therapy with hexyl aminolevulinate induces carbonylation, posttranslational modifications and changed expression of proteins in cell survival and cell death pathways

Photodynamic therapy (PDT) using blue light and the potent precursor for protoporphyrin IX, hexyl aminolevulinate (HAL), has been shown to induce apoptosis and necrosis in cancer cells, but the mechanism remains obscure. In the present study, we examined protein carbonylation, expression levels and post-translational modifications in rat bladder cells (AY-27) after PDT with HAL. Altered levels of expression and/or post-translational modifications induced by PDT were observed for numerous proteins, including proteins required for cell mobility, energy supply, cell survival and cell death pathways, by using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Moreover, 10 carbonylated proteins associated with cytoskeleton, transport, oxidative stress response, protein biosynthesis and stability, and DNA repair were identified using immunoprecipitation, two-dimensional gel electrophoresis and MS. Overall, the results indicate that HAL-mediated PDT triggers a complex cellular response involving several biological pathways. Our findings may account for the elucidation of mechanisms modulated by PDT, paving the way to improve clinic PDT-efficacy.

Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro

Abstract. Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT624 induced more apoptosis than HAL-PDT410 and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.