ALA Concentration Research Articles

Flow Cytometry-Based Photodynamic Diagnosis with 5-Aminolevulinic Acid for the Detection of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is the cancer of plasma cells. Along with the development of new and effective therapies, improved outcomes in patients with multiple myeloma have increased the interest in minimal residual disease (MRD) monitoring. However, the considerable heterogeneity of immunophenotypic and molecular markers of myeloma cells has limited its clinical application. 5-Aminolevulinic acid (ALA) is a natural compound in the heme biosynthesis pathway. Following ALA treatment, tumor cells preferentially accumulate porphyrins because of the differential activities of aerobic glycolysis, known as Warburg effect. Among various porphyrins, protoporphyrine IX is a strong photosensitizer; thus, ALA-based photodynamic diagnosis has been widely used in various solid cancers. Here, the feasibility of flow cytometry-based photodynamic detection of MRD was tested in multiple myeloma. Among various human cell lines of hematological malignancies, including K562 erythroleukemia, Jurkat T-cell leukemia, Nalm6 pre-B cell leukemia, KG1a myeloid leukemia, and U937 monocytic leukemia, human myeloma cell line, KMS18, and OPM2 abundantly expressed ALA transporters, such as SLC36A1 and SLC15A2, and 1 mM ALA treatment for 24 h resulted in nearly 100% porphyrin fluorescence expression, which could be competitively inhibited by ALA transport with gamma-aminobutyric acid. Titration studies revealed that the lowest ALA concentration required to achieve nearly 100% porphyrin fluorescence in KMS18 cells was 0.25 mM, with an incubation period of 2 h. Under these conditions, incubation of primary peripheral blood mononuclear cells resulted in only 1.8 % of the cells exhibiting porphyrin fluorescence. Therefore, flow cytometry-based photodynamic diagnosis is a promising approach for detecting MRD in multiple myeloma.

Cytotoxicity of 5-ALA-conjugated bismuth oxidenanoparticles on KB cell line

Introduction: In recent years, bismuth-based nanomaterials have been widely used in medical researches as imaging, drug delivery and x-ray radiosensitizing agents. Due to their anti-microbial effects against Helicobacter pylori (HP), bismuth colloidal compounds are used to treat various types of diseases such as chronic gastritis. Despite their advantages, bismuth-based compounds have shown many toxic effects in humans. There are few studies on cytotoxicity effects of bismuth (ΙΙΙ) oxide (Bi2O3) nanoparticles (NPs). Based on the natural high affinity of folate for the folate receptor protein (FR), which is commonly expressed on the surface of many human cancers, folate-drug conjugates bind tightly to the FR and trigger cellular uptake via endocytosis. Therefore, we aimed to investigate the cytotoxicity effects of 5-ALA-conjugated Bi2O3 NPs, 5-ALA-folate-conjugated Bi2O3 NPs and free 5-ALA on oral squamous carcinoma cell line (KB cells) for the first time. 5-ALA is used in photo dynamic detection and photodynamic surgery of cancer. Being a precursor of a photosensitizer, 5-ALA is also used as an add on agent for photodynamic therapy. Materials and Methods: Bi2O3 NPs were synthesized and conjugated with folate and 5- aminolevulinic acid (5-ALA). KB cells were cultured monolayer and incubated with 10, 20, 50, 100 and 200 μg/ml concentrations of free 5-ALA, 5-ALA-folate-conjugated Bi2O3 NPs and 5-ALA-conjugated Bi2O3 NPs for 24 hours. The survival of the cells obtained after 24 h of incubation with the materials using MTT assay. Results: Bi2O3 NPs were synthesized (~20 nm) and conjugated to folate and (5-ALA). Results indicated that the significant cellular death were noted at the concentrations more than of 50 μg/ml for all compounds that cell viability reduced to 37%, 49% and 63% in groups receiving 5-ALA-folate-conjugated NPs, 5-ALA-conjugated Bi2O3 NPs and free 5-ALA, respectively. The cytotoxicity of the materials on KB cells also increased with increasing concentrations of synthesized NPs. Conclusion:5-ALA-folate-conjugated NPs, 5-ALA-conjugated Bi2O3 NPs and free 5-ALA were easily taken up by the cells and showed cytotoxic effects at high concentrations. Folic acid conjugation facilitates selective targeting of the KB cells with the overexpression of the folate receptors and improves internalization of the drugs through receptor-mediated endocytosis

Exogenous 5-aminolevulinic acid pretreatment ameliorates oxidative stress triggered by low-temperature stress of Solanum lycopersicum

Low temperature is an important limiting factor in tomato production in early spring and winter. 5-Aminolevulinic acid (ALA) protects crops against varied abiotic stresses. However, the methodology to precisely use ALA to increase the cold tolerance in tomatoes is still not fully known. We therefore explored the effects of ALA concentration, application period, and dose on membrane lipid peroxidation, antioxidation, photosynthesis, and plant growth in different tomato cultivars (Zhongza No. 9, ZZ and Jinpeng No. 1, JP) at low-temperature stress. Results revealed that low temperature caused plants oxidative damage and growth inhibition in both ZZ and JP plants. The ROS (hydrogen peroxide and superoxide anion) accumulation and membrane lipid peroxidation (malondialdehyde content and the relative electrical conductivity) were more remarkable in JP plants than ZZ plants under low temperature. The catalase (CAT) and ascorbate–glutathione cycle (AsA–GSH) induced by ALA reliably eliminated excessive ROS to maintain the redox balance in both tomato cultivars under low-temperature stress. In AsA–GSH cycle, AsA regeneration was mainly catalyzed by dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR), from dehydroascorbate (DHA) to AsA and monodehydroascorbate (MDA) to AsA in ZZ plants, while AsA regeneration in JP plants was mostly catalyzed by DHAR, from DHA to AsA. The ALA optimum concentration was 25 mg L−1. The tomato plants with five true leaves pretreated with 6 mL ALA were more effective than spraying after cold occurred. In conclusion, the two tomato varieties illustrated different capacities to bear low-temperature stress. And ZZ plants were more tolerant to low temperature than JP plants. Precise ALA pretreatment observably alleviated low temperature induced-damage via CAT and AsA–GSH cycle in both cultivars. The regeneration of AsA in AsA–GSH cycle may be more comprehensive in ZZ plants than JP plants, to better tolerate low-temperature stress.

Topical Application of 5-Aminolevulinic Acid Is Sufficient for Photodynamic Therapy on Vocal Folds.

To evaluate the feasibility of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) for vocal fold leukoplakia. Ex vivo and in vivo. 5-ALA was applied topically as a 20% solution to ex vivo canine vocal folds. The penetration depth and concentrations of 5-ALA in tissue were quantified using frozen sectioning and fluorescamine derivatization after 5-ALA contact incubation or topical spraying. Then, 5-ALA solution was sprayed on leporine vocal folds once, twice, or given systemically in vivo. Protoporphyrin IX (PPIX) location was visualized using fluorescence microscopy, and PPIX concentrations were measured using a fluorescent quantitative method. Hematoxylin and eosin (H&E) staining was performed to visualize the histological changes of vocal folds after PDT for each group. Topical incubation of 15 minutes with 5-ALA achieved a penetration depth of over 2 mm and similar concentrations within the superficial 500 μm of epithelium, compared with longer incubation times. Topical spraying of 5-ALA produced sufficient concentrations in vocal folds, but the retention time is short. An in vivo leporine model showed that laryngeal spraying of 20% 5-ALA induced similar penetration depth and concentrations of PPIX compared to systemic administration of 5-ALA. Two sprays of 20% 5-ALA solution with an interval of 30 minutes are needed to produce complete exfoliation of vocal fold epithelium. Topical PDT with laryngeal spraying of 20% 5-ALA solution achieves sufficient therapeutic effects and is potentially applicable for the treatment of vocal fold leukoplakia. NA Laryngoscope, 129:E80-E86, 2019.

Effects of Exogenous 5-Aminolevulinic Acid and 24-Epibrassinolide on Cd Accumulation in Rice from Cd-Contaminated Soil

High grain-Cd-accumulating rice variety Yongyou 9 was planted in Cd-contaminated farmland in Taizhou City, Zhejiang Province, China to study the effects of 5-aminolevulinic acid (ALA) and 24-epibrassinolide (EBR) on Cd accumulation in brown rice. Results showed that the exogenous ALA and EBR had no significant effects on agronomic traits, soil pH and total Cd content in soil, but had some effects on the available Cd content in soil, and significantly influenced the Cd accumulation in the different parts of rice. Results also showed that 100mg/L exogenous ALA significantly reduced the Cd accumulation in brown rice to blow the food safety standard (0.2mg/kg), and also significantly reduced the Cd contents in the roots and culm of rice. However, 200mg/L exogenous ALA treatment increased the Cd content in brown rice remarkably. In addition, 0.15mg/L EBR treatment increased Cd accumulation in roots, culm, leaves and brown rice notably, whereas 0.30mg/L exogenous EBR treatment reduced the Cd accumulation in brown rice properly, but it was not significant. Therefore, proper concentration of ALA can effectively reduce the Cd accumulation in brown rice, which can be used as an effective technical method for the safe production of rice in Cd polluted farmland.

5-Aminolevulinic Acid-Induced Heavy Metal Stress Tolerance and Underlying Mechanisms in Plants

Plants face different types of biotic and abiotic stresses during their life span. Heavy metal (HM) stress is considered as one of the most challenging and emerging threats to sustainable agricultural development and overall economic yield of various plant species. Increasing levels of HMs in arable soils is a main environmental issue due to their deleterious effects on plant growth and productivity. The exogenous application of different plant growth regulators is a well-known strategy to alleviate the adverse effects of HMs stress on plants. In the present review, the role of 5-aminolevulinic acid (ALA) in the alleviation of HM stress in different plants is elaborated. 5-Aminolevulinic acid is identified as a highly efficient ameliorating agent to sustainably neutralize the harmful effects of abiotic stresses in plants. In particular, the role of ALA has been increasingly recognized in improving plant HM stress-tolerance via ALA-mediated control of principal plant-metabolic processes. However, various underlying mechanisms that unravel ALA-induced plant HM stress-tolerance remain unexplored. The application of ALA on HM-stressed plants improves plant height, root length, chlorophyll pigments, antioxidant enzyme activities, nutrient uptake and soluble protein contents and minimizes ultra-structural damage, oxidative stress and HM uptake. Furthermore, it triggers modification of glutathione reductase, ascorbic acid and GSH contents in HM-stressed plants. The lower concentration of ALA proved to be more beneficial in stress amelioration. The cost-effectiveness and efficiency of ALA in improving growth and production of plants under varying growth conditions is still not clear. Nevertheless, over-accumulation of ALA through genetic manipulation can enhance stress-tolerance in plants which is the key area to be investigated. This review article elaborates the potential role of ALA in HM tolerance and highlights the future research dimensions in the related ambits.

5-Aminolevulinic acid moderates environmental stress-induced bunch wilting and stress markers in date palm

Bunch wilting, a disorder of date palm (Phoenix dactylifera L.) and caused by climatic factors (low relative humidity along with high temperatures), critically damages its production in Iran. In this study, the effects of 5-aminolevulinic acid (5-ALA) as precursor of chlorophyll and elicitor of antioxidants applied to be involve directly or indirectly in stress tolerance mechanisms, bunch wilting severity, and other physiological aspects on 10–12 years old date palm in two locations. 5-ALA concentrations included 0 (control), 200, 240 and 280 ppm, applied as aqueous solution at biweekly intervals from fruit set until the beginning of fruit Kimri stage on leaves around the fruit bunches (for three times). Results showed that the yield of trees and bunch weight increased; hydrolytic enzymes activities in stalk and fruit decreased and activities of peroxidase, superoxide dismutase, and catalase, important enzymes of the antioxidative system were increased. The plants’ redox state changed as identified by decrease in H2O2 and proline of fruits. Microelement concentrations of leaves were changed at damage stage. Perhaps, due to mild environmental conditions in location A, lower bunch wilting and better physiological conditions of fruits recorded compared to location B. Foliar application of 5-ALA resulted to a coordinated action of the antioxidative system, affecting the defense-related enzymes. The treatment caused biosynthesis of chlorophyll and adjustments in H2O2 and proline compositions in leaf, stalk, and fruit. Bunch wilting was alleviated by 240 ppm 5-ALA, moderating the response of tree load to environmental stress conditions.

The Effect of Alpha Lipoic Acid (ALA) on <i>S. cerevisiae </i>Cell Proliferation

This experiment investigates what effect ALA may have on the growth of S. cerevisiae, based on extensive studies that have been conducted previously on the therapeutic capabilities of ALA (α-lipoic acid) in various cancers because of its potent antioxidant and apoptotic effects. It manages this because it serves a significant role in enzymatic processes crucial to energy production in cells. In energy metabolism, ALA is responsible for generating acetyl-CoA from pyruvate, and it thus plays a large role in the PDC (pyruvate dehydrogenase complex), an important link connecting glucose metabolism with the citric acid cycle [1]. It helps to shuttle carbon into aerobic respiration, specifically for converting pyruvate into acetyl-CoA and the formation of NADH and carbon dioxide. The process is regulated by enzymes called PDKs, which serve to catalyze suppression of the PDC when the cellular environment becomes nutrient-limited, or glucose scarce. PDKs are speculated to be able to inactivate the PDC via phosphorylation, thus preserving pyruvate for a different metabolic pathway. It is known that overexpression of PDKs blocks oxidative decarboxylation of pyruvate, so targeting the inhibition of PDKs could upregulate PDC activity [2]. Studies have revealed that PDC inhibition is associated with the mechanisms of many metabolic disorders, even cancer (Stacpoole, 2017). In S.cerevisiae cells, which are model organisms for cancer studies, energy is generated typically anaerobically through alcohol fermentation, until this is impacted and it switches to metabolism controlled by the citric acid cycle. This slows down cell proliferation, so it is predicted that as ALA concentrations are increased, cell proliferation will decrease, as indicated by an average cell count of each culture. After experimentation, results revealed that each ALA concentration, 0.125% (2.37×10-20), 0.25% (1.49×10-16), 0.5% (6.41×10-16), and 1% (9.36×10-18), significantly reduced the cell count of their respective cell culture, as compared to the control of 0% ALA. Also, alcohol test strips revealed that with higher concentrations, the cells switched to aerobic respiration as no alcohol was being produced, so fermentation was stopped. This supports the hypothesis that increasing concentrations of ALA will lead to a decrease in cell proliferation, and may indicate that ALA upregulates PDC activity via inhibition of PDKs, which makes it a potential candidate in adjuvant therapies for cancer treatment.

Effects of aminolevulinic acid-based photodynamic therapy on the Toll-like receptor 2 signaling pathway in the murine macrophage line RAW264.7

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of Toll-like receptor 2 (TLR2) and downstream signaling pathway molecules, and secretion of cytokines in murine RAW264.7 cells. Methods The RAW264.7 murine macrophages were induced by inactivated Propionibacterium acnes suspension for the establishment of a cell model of inflammation. The cultured RAW264.7 cells were divided into 5 groups: blank control group receiving normal culture followed by the treatment with phosphate buffer saline (PBS) , model group treated with inactivated Propionibacterium acnes suspension followed by the treatment with PBS, and three ALA groups treated with inactivated Propionibacterium acnes suspension followed by the treatment with 0.03, 0.06 and 0.12 mmol/L ALA, respectively, and infrared radiation at a dose of 16 J/cm2. Enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture supernatant of RAW264.7 cells, and Western blot analysis to determine the protein expression of TLR2 and myeloid differentiation factor 88 (MyD88) , as well as p38, c-Jun N-terminal kinase (JNK) , extracellular signal-regulated kinase (ERK) , inhibitor of κB kinase α (IκBα) and their phosphorylated forms (p-p38, p-JNK, p-ERK and p-IκBα) . Results Compared with the blank control group, the model group showed significantly higher levels of TNF-α ([0.34 ± 0.02]ng/L, P 0.05) , but a significantly lower level of IL-6 ([0.07 ± 0.01]ng/L, F= 114.813, P < 0.01) . The protein expression of TLR2, MyD88, p-p38, p-IκBα, p-JNK and p-ERK was all significantly higher in the model group (0.90 ± 0.14, 1.11 ± 0.13, 0.84 ± 0.04, 1.45 ± 0.20, 2.56 ± 0.06, 3.70 ± 0.40) than in the blank control group (all P < 0.01) , and gradually decreased along with the increase of ALA concentration in a dose-dependent manner. Conclusion Photodynamic therapy can suppress the expression of TLR2 in RAW264.7 murine macrophages, and decrease the secretion of cytokines likely by the TLR2 signaling pathway. Key words: Propionibacterium acnes; Acne vulgaris; Aminolevulinic acid; Photochemotherapy; Toll-like receptor 2; Signal transduction

Pathway engineering in Corynebacterium glutamicum S9114 for 5-aminolevulinic acid production.

5-Aminolevulinic acid (ALA) is a non-protein amino acid with a significant potential for cancer treatment and plant stress resistance. Microbial fermentation has gradually replaced the traditional chemical-based method for ALA production, thus increasing the need for high-ALA-producing strains. In this study, we engineered the glutamate producing strain, Corynebacterium glutamicum S9114, for ALA production. To efficiently convert l-glutamate to ALA, hemA and hemL from Salmonella typhimurium and Escherichia coli were tandemly overexpressed. In addition, ncgl1221 encoding a glutamate transporter was deleted to block glutamate secretion and thus improve ALA production. Furthermore, the intrinsic ribosome-binding site (RBS) of hemB was replaced by a relatively weak RBS to reduce the conversion of ALA to porphyrin. Transcriptional and fermentation data confirmed that inactivation of lysE and putP reduced the conversion of glutamate to arginine and proline, which also contribute to ALA production. The final SA14 strain produced 895mg/L concentration of ALA after 72h incubation in a shake flask. This amount was 58-fold higher than that obtained by the parent strain C. glutamicum S9114. The results demonstrate the potential of C. glutamicum S9114 for efficient ALA production and provide new targets for the development of ALA-producing strains.

In-Vitro Use of 5-ALA for Photodynamic Therapy in Pediatric Brain Tumors.

Light irradiation (635 nm) of cells containing protoporphyrin IX (PPIX) after 5- aminolevulinic acid (5-ALA) pretreatment causes cell death via different pathways including apoptosis and necrosis, as previously demonstrated for malignant glioma cells. To elucidate whether various malignant pediatric brain tumors, which have been shown to accumulate PPIX, would also be susceptible to photodynamic therapy (PDT). Medulloblastoma (DAOY, UW228), pNET (PFSK-1), and rhabdoid tumor (BT16) cell lines were incubated with 5-ALA in variable concentrations for 4 h. Consequently, cells were irradiated by 635 nm diode laser light. After 12 h, cell viability was measured by WST-1 testing and these results were compared to control cells incubated with 5-ALA without irradiation or irradiation only without prior incubation with 5-ALA. We demonstrated significant cell death in malignant pediatric tumor cells after incubation with 5-ALA and laser irradiation in comparison to control groups. In all cell lines, we noticed significant cell death above a 5-ALA concentration of 50 μg/ml (P < .05). Neither 5-ALA incubation alone nor irradiation alone caused cell death. DAOY and PFSK cell lines were more susceptible than UW228 and BT16 cells. We conclude that PDT causes cell death with higher PPIX concentrations after exposure to 5-ALA in vitro in accordance to similar studies with glioma cells. This indicates that PDT might be feasible for eliminating brain tumor cells in malignant pediatric brain tumors. Additionally, we noticed a dependency between fluorescence intensity and death rates.

Effect of aminolevulinic acid-based photodynamic therapy on the expression of protein kinase D1 and its phosphorylation sites in a cutaneous squamous cell carcinoma cell line A431

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of protein kinase D1 (PKD1) in a cutaneous squamous cell carcinoma cell line A431, and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 cells. Methods A431 cells were cultured in vitro, and cell counting kit-8 (CCK-8) assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect. A431 cells at exponential growth phase were randomly divided into 4 groups: control group receiving no treatment, ALA group treated with ALA solution alone, PDT group treated with PDT alone, and ALA-PDT group treated firstly with ALA solution and then with PDT. After 12-, 24-, 36- and 48-hour additional culture, CCK-8 assay was conducted to evaluate the cellular proliferation inhibition, and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry. RT-PCR was performed to determine the expression of protein kinase D1 gene (PRKD1) in A431 cells at different time points after the ALA-PDT treatment, and Western blot analysis to measure protein expression of PKD1 and its phosphorylation at Tyr463 (pTyr463) and Ser916 (pSer916) in A431 cells. Results The combi-nation of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect. After 12-, 24-, 36- and 48-hour additional culture, there were significant differences in the proliferation inhibition rate among the 4 groups (F= 39.56, P 0.05) . No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F= 1.53, P > 0.05) , while there were significant differences in the expression of PKD1 and Tyr463-phosphorylated PKD1 among the 4 groups (F= 10.04, 8.27, both P < 0.05) . Additionally, the ALA-PDT group showed significantly lower expression of PKD1 and Tyr463-phosphorylated PKD1 compared with the control group, ALA group and PDT group (all P < 0.05) . Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT, and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation. Key words: Photochemotherapy; Aminolevulinic acid; Neoplasms, Squamous Cell; Protein kinases; Primary cell culture; A431 cell

Effects of 5-aminolevulinic Acid on the Photosynthesis, Antioxidant System, and α-Bisabolol Content of Matricaria recutita

Matricaria recutita is a widely used medicinal plant with broad pharmacological effects, and α-bisabolol is the main active ingredient of this plant. To improve its α-bisabolol content, M. recutita was sprayed with different concentrations (1.0, 2.0,and 4.0 mmol.L−1) of 5-aminolevulinic acid (ALA) or with water as a control to study the effects of ALA treatment on the photosynthesis, antioxidant system, and α-bisabolol content of M. recutita. Results showed that the photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, soluble protein, total amino acids, soluble sugar, and α-bisabolol of M. recutita were significantly increased. Moreover, the activities of superoxide dismutase, peroxidase, and catalase of M. recutita were also enhanced by ALA treatment. Optimal results were obtained when the concentration of ALA was 2.0 mmol.L−1. Results showed that ALA treatment could improve the α-bisabolol content of M. recutita, and the underlying physiological mechanism was analyzed. ALA treatment was an effective measure for improving the medicinal value of M. recutita.

Abstract A63: Combination of nonlethal photosensitization with a new imidazacridine derivate reduces UACC-62 cell viability

Abstract Melanoma is the skin cancer with the lowest incidence, but the highest aggressiveness and mortality rate. It is known that the actual common resistance to chemotherapy in clinical settings reduces the efficiency of treatments, making urgent the search for new molecules and approaches. The 5-aminolevulinic acid-mediated nonlethal photosensitization (ALA-PDT) is currently one of the most promising forms of cancer treatment, because of its benefits such as noninvasiveness and minimum side effects, usually associated with a transient sensitivity when exposed to the light. In the same context, previous work of our group has shown that the acridine nucleus, present in the imidazacridines compounds, has an essential antiproliferative role and inhibits topoisomerases I and II. Based on that, this in vitro study proposed to evaluate the antineoplastic activity of AC05 (C7H11N3OS), an imidazacridine derivate, in combination with ALA-PDT, against UACC-62, SKMEL28, and A375 melanoma lineages. The cytotoxicity of AC05 and ALA-PDT was analyzed in peripheral blood mononuclear cells (PBMC) from healthy volunteers through an MTT assay, aiming to evaluate the selectivity of therapeutic strategies. Then, using the same method, 1x104 cells of each melanoma lineage were plated in 96-well dark plates and maintained in complete medium, at 37°C in a humidified environment with 5% CO2. After 24 hours, each condition was incubated in triplicate as follow: (a) cells; (b) vehicle control (0.1% dimethyl sulfoxide—DMSO); (c) 1µM doxorubicin (a standard drug commonly used at clinical treatment); (d) 1, 25 and 50µM AC05 dissolved in DMSO and complete medium; (e) 250, 1000 and 2000 µM ALA dissolved in complete medium; (f) laser irradiation - 1, 2 and 3 J/cm2; (g) 250µM ALA + 2J/cm2 laser; (h) 1, 25 and 50 µM AC05 + 2J/cm2 laser; (i) 1, 25 and 50 µM AC05 + 250µM ALA + 2J/cm2 laser. Then, 72 hours later, the cells were incubated with 20 µl of 0.5 mg/ml MTT 3-(4,5-dimethylthiazol-2)-2,5-diphenyl tetrazolium bromide salt by well, during 3 hours, when the reaction was stopped by the incubation of 130 µl of 20% sodium dodecyl sulfate by well. The cell survival was determined after 24 hours, through absorbance measurement at 570 nm by a multiple reader. Results were calculated based on three independent experiments from each lineage. AC05 compound and ALA-PDT did not modify the viability (≤50%) of the PBMC (IC50AC05 ≥ 100 µM and ALA-PDT ≥ 2000 µM). Concerning the melanoma lineages, the SKMEL-28 was the most resistant lineage, since even the approaches in combination (i) were not capable of reduce the viability to less than 50%, except the 2000 µM ALA-PDT (e) that was effective alone. In the A375 melanoma, the single approaches (d and e) did not change the cellular survival ((d)IC50AC05 ≥ 50 µM and (e) ALA-PDT ≥ 2000 µM), although together (i) they reduced this parameter to less than 50% of cell viability. Regarding the UACC-62, either the imidazacridine derivate (d) or the ALA-PDT (e), separately, reduced the cellular viability ((d)IC50AC05 = 28.29 ± 0.6 µM and (e)IC50ALA = 1669,. ± 93.57 µM). However, when in combination they demonstrated a 31% of decreasing in the IC50AC05 capable to reduce the cell viability to less than 50%, when compared to their single effects ((i) IC50AC05 = 20.41 ± 4.37 µM and ALA-PDT = 250 µM). As expected, the experimental conditions with the lowest concentration of ALA (g) or the three concentrations of AC05 (h) combined with the photosensitization have not been able to reduce the cell viability to less than 50%. Altogether, the results showed that the association of the chemotherapy with the photosensitization enhanced the effectiveness of the treatment with AC05. Beyond, the work suggests a new therapeutic potential approach to be explored against UACC-62 and A375 melanoma lineages. Citation Format: Renata V. C. Santos, Artur F. S. Barbosa, Mardonny B. O. Chagas, Maria C. A. Lima, Michelly C. Pereira, Ivan R. Pitta, Maira G. R. Pitta, Moacyr J. B. M. Rego. Combination of nonlethal photosensitization with a new imidazacridine derivate reduces UACC-62 cell viability [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A63.

Effect of photodynamic therapy on the proliferation and apoptosis proteins of fibroblasts in keloid

Objective To investigate the effect of ALA-PDT on the proliferation and apoptosis proteins of KFB. Methods Tissue adherent method was used for the culture of KFB in vitro. The cultured KFB was divided into four groups: the experimental group treated with ALA at 4.0 mmol/L concentration for 5 hours were irradiated by red laser with energy density at 64 J/cm2, and KFB survival was observed after ALA-PDT by CCK-8 method, and then the levels of caspase 3, caspase 8 and caspase 9 proteins were detected after irradiated by PDT. Results When the ALA concentration was 4 mmol/L and the laser energy was 64 J/cm2, the inhibitory effect on the cells was strongest in group A. ALA-PDT could obviously increase the activation of caspase 3, caspase 8 and caspase 9 protein levels and increase the levels of expression [(0.17±0.03) vs (0.58±0.01), (0.21±0.02) vs (0.69±0.15), and (0.13±0.15) vs (0.15±0.02), P<0.05], respectively. Conclusions ALA-PDT can obviously inhibit the proliferation of KFB. Concentration and laser energy are important factors affecting cell survival. ALA-PDT can significantly increase the activity of caspases 3, 8 and 9 proteins in KFB. Key words: Photodynamic therapy; Keloid; Fibroblast; Proliferation; Caspase

Optimization of Hnox Protein Production in Escherichia Coli

Hemeproteins carry a variety of different functions in organisms ranging from steroid biosynthesis to respiration, signaling to drug metabolism. In industry, hemeproteins are used for production of drugs such as: pravastatin for lowering cholesterol, progesterone for hormonal treatment of cancers of uterus and cervix, and cortisone used against allergy and inflammation. Hemeproteins can also be used in drug development and biological remediation. The industrial applications of hemeproteins will expand with development of molecular biology and protein design techniques. One of the obstacles to the widespread use of hemeproteins is difficulties in production of high levels of heme bound protein. This study aims to maximize the amount of heme cofactor bound hemeprotein produced. Here, three important factors affecting hemeprotein production in bacteria are examined: induction by isopropyl β-D-1-thiogalactopyranoside (IPTG), δ-aminolevulinic acid (ALA), precursor for heme biosynthesis, and expression temperature. Effects of these factors on production of thermophilic hemeprotein Tt HNOX are investigated. Since ALA is an expensive molecule, optimization of the amount of ALA used is important. The most suitable conditions to produce Tt HNOX is at low temperature, 0.5 mM IPTG and 1 mM ALA. This study concludes that ALA concentration and expression temperature are important in production of heme bound hemeproteins.

Effects of a hypoenergetic diet rich in α-linolenic acid on fatty acid composition of serum phospholipids in overweight and obese patients with metabolic syndrome.

Plant-derived α-linolenic acid (ALA) may exert cardioprotective effects. Dietary ALA can undergo desaturation and elongation to form long-chain ω-3 polyunsaturated fatty acids, but the extent to which this occurs in humans is unclear. The aim of the study was to examine the effects of an energy-restricted diet enriched with ALA on fatty acid composition of serum phospholipids in patients with metabolic syndrome. The present analysis compared the effects of a hypoenergetic diet high in ALA (3.4 g/d) with a control diet low in ALA (0.9 g/d) on fatty acid composition of serum phospholipids in 81 overweight or obese patients with features of metabolic syndrome. After a 26-wk intervention, concentration of ALA in serum phospholipids remained constant in both diet groups. The control group had a significant decrease in serum phospholipid eicosapentaenoic acid concentration, although no significant intergroup difference was observed. Serum phospholipid docosahexaenoic acid concentration significantly decreased to a similar extent with both interventions. Additionally, both interventions significantly decreased serum phospholipid concentrations of palmitic acid, stearic acid, total saturated fatty acids, linoleic acid, total ω-6 and ω-3 polyunsaturated fatty acids, with no effect of diet group on these changes. Compared with the ALA diet, the control diet led to a significant increase in serum phospholipid oleic acid concentration. Daily intake of 3.4 g of ALA during a 26-wk energy-restricted diet did not lead to an enrichment of serum phospholipids with ALA and did not increase eicosapentaenoic acid due to conversion. Additionally, dietary ALA was unable to compensate for a decrease in serum phospholipid docosahexaenoic acid.

Protein 4.1R is Involved in the Transport of 5-Aminolevulinic Acid by Interaction with GATs in MEF Cells.

5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) has been successfully used in the treatment of cancers. However, the mechanism of 5-ALA transportation into cancer cells is still not fully elucidated. Previous studies have confirmed that the efficiency of 5-ALA-PDT could be affected by the membrane skeleton protein 4.1R. In this study, we investigated the role of 4.1R in the transport of 5-ALA into cells. Wild-type (4.1R+/+ ) and 4.1R gene knockout (4.1R-/- ) mouse embryonic fibroblast (MEF) cells were incubated with 1mm 5-ALA and different concentrations of specific inhibitors of GABA transporters GAT (1-3). Our results showed thatthe inhibition of GAT1 and GAT2 in particular markedly attenuated the intracellular PpIX production, reactive oxygen species (ROS) level and 5-ALA-induced photodamage. However, the inhibition of GAT3 did not show such effects. Further research showedthat 4.1R-/- MEF cells had a lower expression of GAT1 and GAT2 than 4.1R+/+ MEF cells. Additionally, 4.1R directly bound to GAT1 and GAT2. Taken together, GAT1 and GAT2 transporters are involved in the uptake of 5-ALA in MEF cells. 4.1R plays an important role in transporting 5-ALA into cells via at least partly interaction with GAT1 and GAT2 transporters in 5-ALA-PDT.

5-Aminolevulinic acid-based photodynamic therapy of chordoma: In vitro experiments on a human tumor cell line

BackgroundChordomas are very rare tumors of the skull base and the sacrum. They show infiltrating and destructive growth and are known to be chemo- and radio-resistant. After surgical resection, the recurrence rate is high and overall survival limited. As current adjuvant treatments are ineffective, new treatment concepts are urgently needed. 5-aminolevulinic acid-based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. However, it is unknown so far, whether chordomas accumulate protoporphyrin IX (PPIX) after application of 5-ALA and whether they are sensitive to subsequent 5-ALA based PDT. MethodsThe immortalized human chordoma cells U-CH2 were used as in vitro model. After incubation for 4h or 6h with different 5-ALA concentrations, PPIX accumulation was determined by flow cytometry. To assess sensitivity to PDT, chordoma cells were incubated at 30.000cells/well (high cell density) or 15.000cells/well (low cell density) with graded doses of 5-ALA (0–50μg/ml) in 96-well plates and subsequently exposed to laser light of 635nm wavelength (18.75J/cm2). Cell survival was measured 24h after exposure to laser light using the WST-1 assay. ResultsU-CH2 cells dose-dependently accumulated PPIX (ANOVA; p<0.0001). PPIX fluorescence was significantly higher, when cells were incubated with 5-ALA for 6h compared to 4h at higher 5-ALA concentrations (ANOVA/Bonferroni; p≤0.05 for≥30μg/ml 5-ALA). For both cell densities, a 5-ALA dose-dependent decline in viability was observed (ANOVA; p<0.0001). Viability was significantly lower at higher 5-ALA concentrations, when 30.000 cells/wells were treated compared to 15.000cells/well (ANOVA/Bonferroni; p≤0.001 for≥30μg/ml 5-ALA). LD50 was 30.25μg/ml 5-ALA. ConclusionThe human UCH-2 cell line was a very useful in vitro model to study different effects of 5-ALA based PDT. For the first time, it could be shown that human chordoma cells may be destroyed by 5-ALA/PDT.

Amino acid composition of rumen bacteria and protozoa in cattle

Because microbial crude protein (MCP) constitutes more than 50% of the protein digested in cattle, its AA composition is needed to adequately estimate AA supply. Our objective was to update the AA contributions of the rumen microbial AA flowing to the duodenum using only studies from cattle, differentiating between fluid-associated bacteria (FAB), particle-associated bacteria (PAB), and protozoa, based on published literature (53, 16, and 18 treatment means were used for each type of microorganism, respectively). In addition, Cys and Met reported concentrations were retained only when an adequate protection of the sulfur groups was performed before the acid hydrolysis. The total AA (or true protein) fraction represented 82.4% of CP in bacteria. For 10 AA, including 4 essential AA, the AA composition differed between protozoa and bacteria. The most noticeable differences were a 45% lower Lys concentration and 40% higher Ala concentration in bacteria than in protozoa. Differences between FAB and PAB were less pronounced than differences between bacteria and protozoa. Assuming 33% FAB, 50% PAB, and 17% of protozoa in MCP duodenal flow, the updated concentrations of AA would decrease supply estimates of Met, Thr, and Val originating from MCP and increase those of Lys and Phe by 5 to 10% compared with those calculated using the FAB composition reported previously. Therefore, inclusion of the contribution of PAB and protozoa to the duodenal MCP flow is needed to adequately estimate AA supply from microbial origin when a factorial method is used to estimate duodenal AA flow. Furthermore, acknowledging the fact that hydrolysis of 1 kg of true microbial protein yields 1.16 kg of free AA substantially increases the estimates of AA supply from MCP.