Mechanistic studies on δ-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. © 2003 Cancer Research UK.Fil:Correa García, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Casas, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Perotti, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Batlle, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Bermúdez Moretti, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

δ-Aminolevulinic acid transport in murine mammary adenocarcinoma cells is mediated by beta transporters

δ-aminolevulinic acid, the precursor of porphyrin biosynthesis has been used to induce the endogenous synthesis of the photosensitiser protoporphyrin IX for photodynamic therapy in the treatment of various tumours. The aim of this work was to characterise the δ-aminolevulinic acid transport system in the murine mammary adenocarcinoma cell line LM3 using 14C-δ-aminolevulinic acid, to finally improve δ-aminolevulinic acid incorporation in mammalian cells. Our results showed that δ-aminolevulinic acid is incorporated into these cells by two different mechanisms, passive diffusion which is important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in δ-aminolevulinic acid incorporation is a BETA transporter, probably GAT-2

Early neoplastic and metastatic mammary tumours of transgenic mice detected by 5-aminolevulinic acid-stimulated protoporphyrin IX accumulation

A photodynamic technique for human breast cancer detection founded upon the ability of tumour cells to rapidly accumulate the fluorescent product protoporphyrin IX (PpIX) has been applied to transgenic mouse models of mammary tumorigenesis. A major goal of this investigation was to determine whether mouse mammary tumours are reliable models of human disease in terms of PpIX accumulation, for future mechanistic and therapeutic studies. The haeme substrate 5-aminolevulinic acid (5-ALA) (200 mg kg−1) was administered to mouse strains that develop mammary tumours of various histological subtypes upon expression of the transgenic oncogenes HRAS, Polyoma Virus middle T antigen, or Simian Virus 40 large T antigen in the mammary gland. Early neoplastic lesions, primary tumours and metastases showed consistent and rapid PpIX accumulation compared to the normal surrounding tissues, as evidenced by red fluorescence (635 nm) when the tumours were directly illuminated with blue light (380–440 nm). Detection of mouse mammary tumours at the stage of ductal carcinoma in situ by red fluorescence emissions suggests that enhanced PpIX synthesis is a good marker for early tumorigenic processes in the mammary gland. We propose the mouse models provide an ideal experimental system for further investigation of the early diagnostic and therapeutic potential of 5-ALA-stimulated PpIX accumulation in human breast cancer patients

Early detection of urothelial premalignant lesions using hexaminolevulinate fluorescence cystoscopy in high risk patients

<p>Abstract</p> <p>Background</p> <p>To evaluate fluorescence cystoscopy with hexaminolevulinate (HAL) in the early detection of dysplasia (DYS) and carcinoma in situ (CIS) in select high risk patients.</p> <p>Methods</p> <p>We selected 30 consecutive bladder cancer patients at high risk for progression. After endoscopic resection, all patients received (a) induction BCG schedule when needed, and (b) white light and fluorescence cystoscopy after 3 months. HAL at doses of 85 mg (GE Healthcare, Buckinghamshire, United Kingdom) dissolved in 50 ml of solvent to obtain an 8 mmol/L solution was instilled intravesically with a 12 Fr catheter into an empty bladder and left for 90 minutes. The solution was freshly prepared immediately before instillation. Cystoscopy was performed within 120 minutes of bladder emptying. Standard and fluorescence cystoscopy was performed using a double light system (Combilight PDD light source 5133, Wolf, Germany) which allowed an inspection under both white and blue light.</p> <p>Results</p> <p>The overall incidence was 43.3% dysplasia, 23.3% CIS, and 13.3% superficial transitional cell cancer. In 21 patients, HAL cystoscopy was positive with one or more fluorescent flat lesions. Of the positive cases, there were 4 CIS, 10 DYS, 2 association of CIS and DYS, 4 well-differentiated non-infiltrating bladder cancers, and 1 chronic cystitis. In 9 patients with negative HAL results, random biopsies showed 1 CIS and 1 DYS. HAL cystoscopy showed 90.1% sensitivity and 87.5% specificity with 95.2% positive predictive value and 77.8% negative predictive value.</p> <p>Conclusion</p> <p>Photodynamic diagnosis should be considered a very important tool in the diagnosis of potentially evolving flat lesions on the bladder mucosa such as DYS and CIS. Moreover, detection of dysplasic lesions that are considered precursors of CIS may play an important role in preventing disease progression. In our opinion, HAL cystoscopy should be recommended in the early follow-up of high risk patients.</p

ALA and ALA hexyl ester induction of porphyrins after their systemic administration to tumour bearing mice

The use of synthetic lipophilic molecules derived from 5-aminolevulinic acid (ALA) is currently under investigation to enhance cellular ALA penetration. In this work we studied the effect of systemic administration to mice of the hexyl ester of ALA (He-ALA) on porphyrin tissue synthesis as compared to ALA. In most normal tissues as well as in tumour, He-ALA induced less porphyrin synthesis than ALA after its systemic administration either intravenous or intraperitoneal, although explant organ cultures exposed to either ALA or He-ALA revealed equally active esterases. The only tissue that accumulated higher porphyrin levels from He-ALA (seven times more than ALA) was the brain, and this correlated well with a rapid increase in ALA/He-ALA content in brain after administration of He-ALA. This may be ascribed to a differential permeability to lipophilic substances controlled by the blood–brain barrier, a feature which could be further exploited to treat brain tumours

Hypoxia significantly reduces aminolaevulinic acid-induced protoporphyrin IX synthesis in EMT6 cells

We have studied the effects of hypoxia on aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) synthesis in EMT6 monolayer cultures characterized by different cell densities and proliferation rates. Specifically, after ALA incubation under hypoxic or normoxic conditions, we detected spectrofluorometrically the PpIX content of the following populations: (a) low-density exponentially growing cells; (b) high-density fed-plateau cells; and (c) high-density unfed-plateau cells. These populations were selected either for the purpose of comparison with other in vitro studies (low-density exponentially growing cells) or as representatives of tumour regions adjacent to (high-density fed-plateau cells) and further away from (high-density unfed-plateau cells) capillaries. The amount of PpIX per cell produced by each one of these populations was higher after normoxic ALA incubation. The magnitude of the effect of hypoxia on PpIX synthesis was dependent on cell density and proliferation rate. A 42-fold decrease in PpIX fluorescence was observed for the high-density unfed-plateau cells. PpIX production by the low-density exponential cells was affected the least by ALA incubation under hypoxic conditions (1.4-fold decrease), whereas the effect on the high-density fed-plateau population was intermediate (20-fold decrease). © 1999 Cancer Research Campaig