Intracellular ALA Research Articles

Aminolevulinic acid dendrimers in photodynamic treatment of cancer and atheromatous disease.

The use of endogenous protoporphyrin IX after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). We have previously reported that the conjugation of ALA dendrimers enhances porphyrin synthesis. The first aim of this work was to evaluate the ability of ALA dendrimers carrying 6 and 9 ALA residues (6m-ALA and 9m-ALA) to photosensitise cancer cells. For this aim, we employed LM3 mammary carcinoma cells. In these tumour cells, at low concentrations porphyrin synthesis from dendrimers was higher compared to ALA, whereas at high concentrations, porphyrin synthesis was similar from both compounds. Topical application of ALA dendrimers on the skin overlying a subcutaneous LM3 implanted tumour showed no diffusion of the molecules either to distant skin sites or to the adjacent tumour, suggesting a promising use of the ALA macromolecules in superficial cancer models. As a second objective, we proposed the use of ALA-dendrimers in vascular PDT for the treatment of atherosclerosis. Thus, we focused our studies on ALA-dendrimer's selectivity towards macrophages in comparison with endothelial cells. For this aim we employed Raw 264.7 macrophages and HMEC-1 microvasculature cells. Porphyrin synthesis induced in macrophages by 6m-ALA and 9m-ALA (3 h, 0.025 mM) was 6 and 4.6 times higher respectively compared to the endothelial cell line, demonstrating the high affinity of ALA dendrimers for macrophages. On the other hand, ALA employed at low concentrations was slightly selective (1.7-fold) for macrophages. Inhibition studies suggested that ALA dendrimer uptake in macrophages is mainly mediated by caveloae-mediated endocytosis. Our main conclusion is that in addition to being promising molecules in PDT of superficial cancer, ALA dendrimers may also find applications in vascular PDT, since in vitro they showed selectivity to the macrophage component of the atheromatous plaque, as compared to the vascular endothelium.

Improved 5-Aminolevulinic Acid Production with Recombinant Escherichia coli by a Short-term Dissolved Oxygen Shock in Fed-batch Fermentation

5-Aminolevulinic acid (ALA) is a common precursor for tetrapyrrole compounds in all kinds of organisms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen (DO) shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombinant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase (ALAS) activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 min in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 min. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g·L−1 (72 mmol·L−1), which is the highest yield in the fermentation broth reported up to now.

Clearance mechanism of protoporphyrin IX from mouse skin after application of 5-aminolevulinic acid

5-Aminolevulinic acid (ALA) or its esters mediated photodynamic therapy (PDT) is the most widely practiced form of PDT in dermatology. One of its advantages is that undesirable photosensitization lasts only for 24-48 h. In order to optimize ALA-PDT it is necessary to understand the mechanisms of intracellular production and clearance of PpIX (efflux from cells into blood stream and/or its conversion into haem). The aim of this study is to investigate the factors controlling the clearance of intracellular PpIX from healthy skin of mice. PpIX was induced in mouse skin by topical or systemic application of ALA or by topical application of the iron chelator ethylenediaminetetraacetic acid (EDTA). Fluorescence spectroscopy was used to study PpIX kinetics in alive and dead skin. Topical application of ALA or EDTA leads to porphyrin production in living skin, but not in excised skin. The clearance rates of PpIX from alive and dead skin were the same in the absence of an intracellular ALA pool. The clearance half-life of EDTA-induced PpIX was 4-7 times longer than that of PpIX after application of ALA. Skin temperature and intracellular iron availability strongly affect PpIX clearance, while ALA application mode (topical versus systemic) and skin viability (dead versus alive) have no influence on PpIX decay. These results demonstrate that the clearance kinetics of PpIX from skin are determined mostly by the conversion of PpIX into haem, while the cellular efflux of PpIX into blood plays a minor role.

Effect of Gene Amplifications in Porphyrin Pathway on Heme Biosynthesis in a Recombinant Escherichia coli

A recombinant E. coli co-expressing ALA synthase (hemA), NADP-dependent malic enzyme (maeB), and dicarboxylic acid transporter (dctA) was reported to synthesize porphyrin derivatives including iron-containing heme. To enhance the synthesis of bacterial heme, five genes of the porphyrin biosynthetic pathway [pantothenate kinase (coaA), ALA dehydratase (hemB), 1-hydroxymethylbilane synthase (hemC), uroporphyrinogen III synthase (hemD), and uroporphyrinogen III decarboxylase (hemE)] were amplified in the recombinant E. coli co-expressing hemA-maeB-dctA. Pantothenate kinase expression enabled the recombinant E. coli to accumulate intracellular CoA. Intracellular ALA was the most enhanced by uroporphyrinogen III synthase expression, porphobilinogen by ALA dehydratase expression, and uroporphyrin and coproporphyrin by 1- hydroxymethylbilane synthase expression. The strain coexpressing coaA, hemA, maeB, and dctA produced heme of 0.49 micromol/g-DCW, which was twice as much from the strain without coaA expression. Further strain improvement for the porphyrin derivatives is discussed based on the results.

Effect of surfactant on 5-aminolevulinic acid uptake and PpIX generation in human cholangiocarcinoma cell

Photodynamic therapy (PDT) is a palliative therapy and has been used to cure cholangiocarcinoma (CC), which has a poor prognosis and limited available curative therapy. PDT was shown to improve the median survival time of advanced-stage patients. Recently, 5-aminolevulinic acid (ALA) has been used as a pro-photosensitizer, which can be transferred to intercellular protoporphyrin IX (PpIX), which is a strong photosensitizer, via the heme pathway. The main limitation of using ALA in PDT is the hydrophilic properties of ALA, which results in low cellular uptake. In this study, non-ionic surfactants, pluronic F68 (PF68) and Tween 80 (TW80), were used to address this limitation. The human CC cell line, HuCC-T1, was cotreated with ALA and different concentrations of surfactants for 4h. The effect of surfactants was evaluated by monitoring the uptake of ALA, the fluorescence intensity of PpIX, and the cell survival rate after suitable light irradiation. Cotreatment with the surfactant resulted in an increased intracellular ALA level, PpIX formation, and phototoxicity.

Influence of culture conditions on the production of extra-cellular 5-aminolevulinic acid (ALA) by recombinant E. coli

Extra-cellular production of 5-aminolevulinic acid (ALA) by recombinant E. coli harboring a fusion gene hemA:egfp was investigated in shake flasks and a bioreactor with chemically defined media by varying physiological factors such as the concentrations of precursors (glycine and succinic acid), and levulinic acid (LA). Optimum concentrations of glycine and succinic acid were found to be 15 and 30 mM, respectively. When 30 mM LA was added to the culture medium at the end of the exponential cell growth phase, intra-cellular ALA dehydratase activity was suppressed. Some imbalances might be caused by the addition of titrants to the cultivation. Therefore, the recombinant E. coli used in this study were grown with no control over the culture pH in the beginning phase. The recombinant E.coli produced significant ALA in the initial pH ranges of 6.1 and 6.5, i.e. without the addition of titrants. IPTG induction for the expression of the fusion gene did not enhance the production of extra-cellular ALA. Repeated addition of glycine, succinic acid, and LA increased the production of extra-cellular ALA and the inhibition of intra-cellular ALA activity, with up to 1.3 g/l ALA having been produced in the cultivation.

Mechanistic studies on delta-aminolevulinic acid uptake and efflux in a mammary adenocarcinoma cell line.

δ-aminolevulinic acid (ALA) is the precursor in the biosynthesis of porphyrins. The knowledge of both the regulation of ALA entrance and efflux from the cells and the control of porphyrin biosynthesis is essential to improve ALA-mediated photodynamic therapy. In this work, we studied the regulation of ALA uptake and efflux by endogenously accumulated ALA and/or porphyrins in murine mammary adenocarcinoma cells. Under our set of conditions, the haem synthesis inhibitor succinyl acetone completely prevented porphobilinogen and porphyrin synthesis from ALA, and led to an increase in the intracellular ALA pool. However, neither intracellular ALA nor porphyrin pools regulate ALA uptake or efflux during the first 15 min of the process. Based on temperature dependence data, ALA but not γ-aminobutyric acid (GABA) efflux is mediated by a diffusion mechanism. Moreover, the addition of extracellular GABA not only did not influence the rate of ALA efflux but on the contrary it affected ALA uptake, showing the contribution of a saturable mechanism for the uptake, but not for the efflux of ALA from the cells.

Inhibition of 5-aminolevulinic acid dehydratase in recombinant Escherichia coli using d-glucose

For the overproduction of 5-aminolevulinic acid (ALA) from recombinant Escherichia coli, the inhibition of ALA dehydratase on both small scale by using an eppendorf tube, and on a large scale by using a fermenter, the in vitro glycation and the inactivation of enzymes on the ALA dehydratase under several experimental conditions were investigated. The presence of 0.5–10 mM of d-glucose caused a concentration-dependent inhibition of recombinant E. coli ALA dehydratase activity. The ALA dehydratase levels were dependent on the pH of the medium, with the maximal activities occurring at 8.0. The inhibition constant, K i, of intracellular ALA dehydratase by d-glucose and levulinic acid (LA) were 1.02 and 0.32 mM, respectively. The addition of 10 mM of d-glucose drastically inhibited the ALA dehydratase activity (85% inhibition), in turn, the highest level of extracellular ALA production (3.8 g/l) was achieved. Based upon those results, we concluded that d-glucose decreased the ALA dehydratase activity both by the competitive inhibition with substrate and by the inactivation of enzyme protein, and that the inactivation of ALA dehydratase by d-glucose may require glycation metabolism of d-glucose at least in part.

Porphyrin Biosynthesis Intermediates Are Not Regulating δ-Aminolevulinic Acid Transport in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, as in all eukaryotic organisms, δ-aminolevulinic acid (ALA) is a precursor of porphyrin biosynthesis, a very finely regulated pathway. ALA enters yeast cells through the γ-aminobutyric acid (GABA) permease Uga4. The incorporation of a metabolite into the cells may be a limiting step for its intracellular metabolization. To determine the relationship between ALA transport and ALA metabolization, ALA incorporation was measured in yeast mutant strains deficient in the δ-aminolevulinic acid-synthase, uroporphyrinogen III decarboxylase, and ferrochelatase, three enzymes involved in porphyrin biosynthesis. Results presented here showed that neither intracellular ALA nor uroporphyrin or protoporphyrin regulates ALA incorporation, indicating that ALA uptake and its subsequent metabolization are not related to each other. Thus a key metabolite as it is, ALA does not have a transport system regulated according to its role.

Genetically engineered Rhodobacter sphaeroides for the overproduction of δ-aminolevulinic acid

Genes for β and α polypeptides of the light-harvesting II complexes in Rhodobacter sphaeroides 2.4.1 are encoded by the pucBA operon, and their expression is highly regulated by oxygen level and light intensity. δ-Aminolevulinic acid (ALA) in this bacterium is synthesized by ALA synthase isozymes encoded by hemA and hemT. Recombinant plasmids carrying either a pucB′-hemA transcriptional fusion (pAS608A) or a pucB′-hemT translational fusion (pAS607A) have been constructed. Derivatives of Escherichia coli SASP19 (hemA− mutant), harboring either pAS608A or pAS607A, still required exogenous ALA supplementation. However, each of these functions could be expressed in R. sphaeroides AT1 (hemA− and hemT − mutant of R. sphaeroides 2.4.1) such that AT1 could grow in the medium without exogenous ALA supplementation. Introduction of pAS608A into R. sphaeroides 2.4.1 resulted in the increase of ALA synthase activity under both aerobic and photosynthetic growth conditions, while the introduction of pAS607A resulted in the increase of ALA synthase only under aerobic conditions. The production of extracellular and intracellular ALA in R. sphaeroides 2.4.1 (pAS608A) or R. sphaeroides 2.4.1 (pAS607A) was not significantly different from that of the wild-type strain in either aerobic or photosynthetic growth conditions.

Cinétiques de décarboxylation oxydative extra- et intracellulaire d'α-acétolactate par un Lactococcus lactis ssp. lactis var. diacetylactis

Kinetic studies on œ-acetolactic acid extra and intracellular oxidative decar- boxylation to diacetyl by Laetoeoeeus laetis ssp. /aetis var. diaeetylaetis SD 933. œ-acetolac- tie acid (ALA) oxidative decarboxylation is thought to be the dominantmechanism for the dairy flavour diacetyl production by lactic acid bacteria of Leuconostoc and Lactococcus genera. However, kinetie data on that particular reaction are lacking due to the conjunction of the high reac- tivity of ALA in presence of oxygen and the diacetyl and ALA specifie measurement difficulties. The use of a gas chromatographie technique coupled with a gas membrane sensor equipped bio- reactor with a controlled atmosphere nonetheless allows the on-line acquisition of diacetyl pro- duction kinetics by lactococci cultures or directly from ALA. Thus, this paper provides kinetic data on both diacetyl microbiological production and extra- and intracellular ALA oxidative decar- boxylation. The comparison ofthese kinetics shows that oxidative decarboxylation can account for diacetyl biological production and that this reaction is located at the intracellular level. More- over, the evolution of the pattern of intracellular kineties with and without heat treatment demons- trates the action of a thermolabile compound in oxidative decarboxylation. © InralElsevier, Paris. lactic acid bacteria / Laetoeoeeus / dairy flavour / diacetyl / œ-acetolactate

Haem precursor delta-aminolaevulinic acid induces activation of the cytosolic iron regulatory protein 1.

Control of cellular iron homoeostasis is performed by iron regulatory protein 1 (IRP1) through post-transcriptional modifications. This protein is sensitive to intracellular iron availability, being activated at low iron levels and inactivated at high iron levels, conditions that signal the increased expression of the transferrin receptor or of ferritin respectively. IRP1 is known to be activated by some oxidants such as H2O2 and NO. delta-Aminolaevulinic acid (ALA), previously found to produce reactive oxygen species and a carbon-centred radical, to release iron from ferritin, and to increase rat liver and brain non-haem iron and ferritin, was investigated for its effects on IRP1 activity in cultured hamster pulmonary fibroblasts. We have found that 1-2 mM ALA produced a 2-3-fold activation of IRP. On incubation with 1-4 mM succinylacetone methyl ester, a potent ALA dehydratase inhibitor, a 3-4-fold activation of the protein was observed, accompanied by a 40% increase in the intracellular ALA concentration. When cells were incubated in the presence of ALA or succinylacetone methyl ester, N-acetylcysteine inhibited IRP1 activation, suggesting that the observed effect is mediated by an oxidative process. We surmise that ALA-induced IRP1 activation might act as a co-sensor of iron homoeostasis.

Inhibition of 5-aminolevulinic acid (ALA) dehydratase by undissociated levulinic acid during ALA extracellular formation by Rhodobacter sphaeroides

The inhibition of ALA dehydratase by levulinic acid during ALA extracellular formation of Rhodobacter sphaeroides correlated with the concentration of undissociated form of levulinic acid irrespective of culture pH. The inhibition constant, Ki, of intracellular ALA dehydratase by Dixon plots was 2.95 μM. Undissociated levulinic acid therefore functions as an inhibitor of ALA dehydratase.

The role of ALA‐S and ALA‐D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae

Catabolite repression and derepression on delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R+) were investigated. ALA-S and ALA-D activities and intracellular ALA (I-ALA) at different physiological states of the cells were measured. In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed. In YPEt medium, however, both ALA-S and ALA-D activities were higher than in YPD, but the I-ALA content and the enzymic activity profiles shown by the two strains were quite different. It appears, therefore, that the mutation causes a deregulation of ALA-S, so that its activity is kept at a high level throughout the cell cycle. This would explain the increased levels of cytochromes present in the mutant. This mutation may affect some regulatory aspect of ALA formation and renders an ALA-S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.

Production of 5-aminolevulinic acid by photosynthetic bacteria

Extracellular formation of 5-aminolevulinic acid (ALA) by adding levulinic acid (LA), an inhibitor of ALA dehydratase, was examined in the anaerobic-light culture of Rhodobacter sphaeroides. The addition of LA (10–25 mmol/ l) during the middle log phase retarded the growth and accelerated the extracellular formation of ALA, while over 50 mmol/ l completely suppressed both growth and formation. The formation of ALA was closely related to intracellular ALA synthetase activity. Light intensity was also an important factor for enhancing ALA formation. The optimal condition, addition of 15 mmol/ l of LA during the middle log phase with 3 klx illumination, resulted in ALA formation of 0.26 mol/ l. In addition, supplementation with glycine (30 mmol/ l) and succinate (30 mmol/ l), precursors of ALA biosynthesis, enhanced ALA formation up to ca. 2 mmol/ l.