Presence Of 5-ALA Research Articles

Development and optimisation of in vitro sonodynamic therapy for glioblastoma

Sonodynamic therapy (SDT) is currently on critical path for glioblastoma therapeutics. SDT is a non-invasive approach utilising focused ultrasound to activate photosensitisers like 5-ALA to impede tumour growth. Unfortunately, the molecular mechanisms underlying the therapeutic functions of SDT remain enigmatic. This is primarily due to the lack of intricately optimised instrumentation capable of modulating SDT delivery to glioma cells in vitro. Consequently, very little information is available on the effects of SDT on glioma stem cells which are key drivers of gliomagenesis and recurrence. To address this, the current study has developed and validated an automated in vitro SDT system to allow the application and mapping of focused ultrasound fields under varied exposure conditions and setup configurations. The study optimizes ultrasound frequency, intensity, plate base material, thermal effect, and the integration of live cells. Indeed, in the presence of 5-ALA, focused ultrasound induces apoptotic cell death in primary patient-derived glioma cells with concurrent upregulation of intracellular reactive oxygen species. Intriguingly, primary glioma stem neurospheres also exhibit remarkably reduced 3D growth upon SDT exposure. Taken together, the study reports an in vitro system for SDT applications on tissue culture-based disease models to potentially benchmark the novel approach to the current standard-of-care.

Triphenylphosphonium conjugated gold nanotriangles impact Pi3K/AKT pathway in breast cancer cells: a photodynamic therapy approach

Although gold nanoparticles based photodynamic therapy (PDT) were reported to improve efficacy and specificity, the impact of surface charge in targeting cancer is still a challenge. Herein, we report gold nanotriangles (AuNTs) tuned with anionic and cationic surface charge conjugating triphenylphosphonium (TPP) targeting breast cancer cells with 5-aminoleuvinic acid (5-ALA) based PDT, in vitro. Optimized surface charge of AuNTs with and without TPP kill breast cancer cells. By combining, 5-ALA and PDT, the surface charge augmented AuNTs deliver improved cellular toxicity as revealed by MTT, fluorescent probes and flow cytometry. Further, the 5-ALA and PDT treatment in the presence of AuNTs impairs cell survival Pi3K/AKT signaling pathway causing mitochondrial dependent apoptosis. The cumulative findings demonstrate that, cationic AuNTs with TPP excel selective targeting of breast cancer cells in the presence of 5-ALA and PDT.

Dynamics of 5-Aminolevulinic Acid-Induced Accumulation of Protoporphyrin IX in Three Cell Lines of Different Origin

The dynamics of synthesis and accumulation of protoporphyrin IX induced by 5-aminolevulinic acid (5-ALA) was studied using confocal fluorescence microscopy in three established cell lines: immortalized cells originated from human tumor tissues—epithelial carcinoma (HeLa cells) and lung adenocarcinoma (A549 cells)—and immortalized fibroblast-like mouse embryo cells (3T3 line). The accumulation of protoporphyrin IX in cell cultures was estimated by analyzing the intensity of its fluorescence. The dynamics of changes in the fluorescence intensity with the cell incubation duration in the presence of 5-ALA and with the 5-ALA concentration was studied.

Effect of Silver Nanoparticles on Improving the Efficacy of 5-Aminolevulinic Acid-Induced Photodynamic Therapy

Introduction: The most important limitation of 5-aminolevulinic acid (5-ALA)-induced photodynamic therapy (PDT) is the efficacy of the cells in converting 5-ALA to protoporphyrin IX. The present study aimed to investigate the effectiveness of silver nanoparticles (AgNPs) with the photosensitivity at the surface plasmon resonance wavelength on 5-ALA-mediated PDT. Material and Methods: First of all, the toxicity of 5-ALA, AgNPs, and combined 5-ALA and AgNPs was evaluated on DFW cell line derived from melanoma. After choosing the optimal concentration, both pulsed and continuous light irradiations were conducted at different doses using a light‑emitting diode source in the groups receiving 5-ALA, AgNPs, as well as 5-ALA and AgNPs combination, and the controls. The cell survival was evaluated 24 h after irradiation using MTT assay. Results: According to the results, light exposure did not significantly change cell survival in the absence or presence of AgNPs. However, light exposure in the presence of 5-ALA and AgNPs/5-ALA combination caused a significant reduction in the cell survival. The necessary light exposure to induce 50% cell death (ED50) in the presence of 5-ALA was 1280 mJ/cm2; however, this value was 280 mJ /cm2 in the presence of combined AgNPs and 5-ALA. Conclusion: As the findings indicated, PDT had a higher efficacy in the presence of combined 5-ALA and AgNPs than in the sole 5-ALA presence. Nonetheless, further studies are required to evaluate the definitive mechanism of these findings.

Involvement of nitric oxide in 5-aminolevulinic acid-induced antioxidant defense in roots of Elymus nutans exposed to cold stress

Nitric oxide (NO) and 5-aminolevulinic acid (5ALA) play fundamental roles in plant responses to environmental stresses, but their cross-talk in antioxidant defense in cold-stressed Elymus nutans Griseb. have not been investigated. We herein report that 5ALA and NO donor, sodium nitroprusside (SNP), alleviated cold stress-induced plant growth inhibition and lipid peroxidation in roots of two E. nutans ecotypes (Damxung, DX and Zhengdao, ZD). However, application of an NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (PTIO) differentially blocked these protective effects indicating that an inhibition of NO accumulation reduced 5ALA-enhanced cold resistance. Application of exogenous 5ALA or NO markedly up-regulated activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, enhanced reduced glutathione accumulation and reduced glutathione to oxidized glutathione ratio, activated plasma membrane (PM) H+-ATPase, and reduced Na+/K+ ratio in roots of the two E. nutans ecotypes. Moreover, in the presence of 5ALA, nitric oxide synthase (NOS) activity and NO release in cold-resistant DX were higher than those in cold-sensitive ZD. Conversely, both NO treatment and inhibition of endogenous NO accumulation by PTIO or NOS inhibitor Nω-nitro-L-arginine did not induce 5ALA production. These results suggest that NO might be acting as a downstream signal to mediate 5ALA-induced cold resistance by activating antioxidant defense and PM H+-ATPase and maintaining Na+ and K+ homeostasis.

Comparative study of X-ray treatment and photodynamic therapy by using 5-aminolevulinic acid conjugated gold nanoparticles in a melanoma cell line

Purpose: The most important challenges in photodynamic therapy (PDT) are related to the limited penetration of light and the low uptake of photosensitizers. In radiotherapy, they are correlated to radiation damage of normal tissues. Therefore, a targeted radio and photosensitizer can reduce the limitations of the mentioned methods. In this study, photosensitizing and radio-sensitizing effects of 5-aminolevulinic acid (5ALA)-conjugated GNPs were investigated. Materials and methods: First, cell toxicities of 5ALA, GNPs and a conjugate were assessed on Mel-Rm cell line. Then, the radio sensitizing effect of every agent was studied. Different experiments were designed in four separate groups, each group containing six subgroups receiving different radiation doses by using a superficial X-ray tube. Furthermore, the photosensitizing efficacy of the agents was evaluated after cells were irradiated by a He–Ne laser at four light doses in separate groups. Results: With regards to radio sensitivity assessments, there was no significant difference between different irradiation doses. The investigation on photosensitivity of 5ALA and a conjugate showed significant differences between the control (without illumination) and groups that received PDT in the presence of 5ALA and conjugate, wherein ED50 were estimated at 136.2 J/cm2 and 56.2 J/cm2, respectively. With regards to PDT experiments, the conjugate induces cell death more than twice in comparison with 5ALA. Conclusion: The conjugate does not cause any enhancement of radiation efficiency on MeL-Rm cell line. With regards to PDT, we found that the conjugate induced cell death at twice the rate when compared with 5ALA alone. Therefore, the conjugate can be an appropriate delivery agent for 5ALA and may also enhance the destruction of tumor cells. Finally, comparing the two types of treatment shows that PDT is a more efficient treatment for this cell line.

Experimental Study to Understand Nonspecific Protoporphyrin IX Fluorescence in Brain Tissues Near Tumors After 5-Aminolevulinic Acid Administration

(PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA), which appears in various tumors including malignant gliomas, is a good navigator for tumor resection. However, some non-neoplastic tissue also shows a weak PpIX fluorescence. The origin of non-neoplastic PpIX is still unknown, and three hypotheses on its origins can be proposed: tumor cell-derived PpIX followed by dispersal via bulk flow, direct PpIX production by normal brain or gliotic brain tissue, or PpIX produced in other organs leaking from the tumor vessels. We investigated the possibility of these three origins experimentally. In vitro, we exposed various cultured glioma and meningioma cell lines to different conditions of 5-ALA. After this, the degree of fluorescence of the culture medium and cells was each quantitatively measured and analyzed by means of photometrical assay. We administered 5-ALA directly into the normal brains of rats by using convection-enhanced delivery technique, and we also administered 5-ALA or PpIX systemically after blood-brain barrier (BBB) disruption. As a result of our in vitro study using cell lines, we found fluorescence of extracellular PpIX in the culture medium. As a result of rat study, we found PpIX fluorescence of the extracted normal brain after systemic administration of 5-ALA with BBB disruption. However, no PpIX fluorescence was observed when we administered PpIX. It was demonstrated that PpIX was quickly excreted by tumor cells out to the extracellular space, and that even normal brain tissue can produce PpIX in the presence of 5-ALA.

Raman spectroscopy of bladder tissue in the presence of 5-aminolevulinic acid

Raman spectroscopy has the ability to provide differential diagnosis of different cancers with high sensitivity and specificity. A major limitation in its clinical application is the weak nature of Raman signal, which inhibits scanning large surface areas of tissues. In bladder cancer diagnosis, fluorescence-guided endoscopy with 5-aminolevulinic acid (5-ALA) has gained interest as a technique that can provide such spatial differentiation, thus improving early detection and more complete removal of superficial tumors. However, several studies have demonstrated the poor specificity of this modality. Combining fluorescence with Raman spectroscopy could improve its diagnostic capability. However, little is known about the effect of agents such as 5-ALA on Raman spectra of tissue. In this paper, we present measuring Raman spectroscopy from benign and malignant bladder tissues in the presence of 5-ALA and attempt to evaluate the potential to discriminate between different pathologies. Raman spectra were recorded from 92 bladder biopsies without 5-ALA and 38 biopsies with 5-ALA using a Raman microspectrometer system at 830 nm excitation. Empirical and multivariate statistical techniques were used for data analysis. Algorithms were developed to determine the effect of 5-ALA on tissue and its influence on the prediction ability of a preliminary benign/malignant prediction model. In samples with 5-ALA, an overall decrease in Raman intensity was observed when compared to the Raman spectra from samples without 5-ALA. Additionally, differences in relative intensities at 1270 and 1330 cm −1 were also noted. However, significant differences were observed in the Raman spectra of benign and malignant samples with 5-ALA indicating the potential of using Raman spectroscopy for discriminating bladder cancer in the presence of 5-ALA. The Principal-Component fed Linear-Discriminant Analysis (PCA/LDA) algorithm derived from biopsies in the absence of 5-ALA used to predict biopsies in the presence of 5-ALA resulted in an overall sensitivity and specificity of 42.6% and 71.1%, respectively. This suggests the presence of 5-ALA in tissue affects the Raman spectra. A PCA/LDA algorithm based on fluorescence information (i.e. PpIX fluorescence positive or negative) and the Raman spectrum of 5-ALA biopsies, had a sensitivity and specificity of 100% and 80.8%, respectively. This study demonstrates that applying 5-ALA affects the Raman spectra of bladder tissues. However, benign/malignant differentiation can be accomplished with a preliminary PCA/LDA algorithm, suggesting the potential of a combined diagnostic modality in vivo.

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

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 mMβ-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.

5-aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters.

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 (K(m) = 8-10 mM and Vmax = 18-20 nmol.(mg protein x h)-1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32 degrees C. The activation energy was 112 kJ.mol-1 in the temperature range 15 degrees-32 degrees C and 26 kJ.mol-1 above 32 degrees 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 beta-amino acids, beta-alanine and taurine and by gamma-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-beta-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM beta-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 microM) was not significantly influenced by beta-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by beta-amino acid and GABA carriers in this cell line.

Induction of cytochrome P450 activities by polychlorinated biphenyls in isolated mouse hepatocytes: Influence of Ah-phenotype and iron

Exposure of cultured primary hepatocytes from Ah-responsive male C57BL/10ScSn mice to a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) at 0.1–20 μg/mL for up to 96 hr induced cytochrome P4501AI-mediated activity (ethoxyresorufin 0-deethylase, EROD) up to 50-fold. In contrast, pentoxyresorufin O-dealkylase (PROD), which in some circumstances is a measure of phenobarbitone induced cytochrome P450 isoenzymes, was induced only 5-fold. There were similar findings on EROD activities with the pure compounds 3,3′,4,4′,5,5′-hexachlorobiphenyl, 3,3′,4,4′,5,5′-hexabromobiphenyl and 3,3′,4,4′-tetrachlorobiphenyl(TCB) and also β-naphthoflavone but not with 2,2′,4,4′-TCB or phenobarbitone. The higher concentrations of Aroclor 1254 were also associated with cytotoxicity as estimated by release of alanine aminotransferase (ALT) into the medium. Unlike in C57BL/10ScSn hepatocytes induction of EROD and cytotoxicity was minimal in hepatocytes from the Ah-non-responsive strain DBA/2. Although in vivo the hepatic toxicity and carcinogenicity of polyhalogenated aromatics are markedly potentiated by iron, no enhancement of the cytotoxicity of Aroclor 1254 towards C57BL/10ScSn hepatocytes by iron was observed in vitro. However, iron caused decreased EROD activities and possibly cytochrome P4501AI (as judged by Western blotting) as in vivo. Even in the presence of iron and the haem precursor 5-aminolaevulinic acid (5-ALA) there was no development of uroporphyria in this system although this occurs with Aroclor in vivo and is enhanced by iron. Accumulation of uroporphyrin did occur after extended culture of C57BL/10ScSn hepatocytes on matrigel for 8 days in the presence of 5-ALA and Aroclor 1254 but again no potentiation by iron was observed. Thus, although culture of Ah-responsive and -non-responsive hepatocytes mimics some aspects of the mechanisms of in vivo toxicity of PCBs, there is some unknown associated influence of iron metabolism which cannot, as yet, be produced vitro but which is of importance in vivo.