The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank's balanced salt solution (HBSS; n = 5), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group ( P < 0.05) and the αMEM group ( P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l -1 , 2 mmol l -1 , and 5 mmol l -1 ) to determine the most effective concentration of 5-ALA. The 2 mmol l -1 5-ALA group ( n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l -1 , and 5 mmol l -1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control ( n = 3) and 2 mmol l -1 5-ALA ( n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes ( n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l -1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = -1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l -1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.
Male ; Animals ; Testis/cytology* ; Aminolevulinic Acid/pharmacology* ; Mice ; Organ Preservation/methods* ; Organ Preservation Solutions/pharmacology* ; Cryopreservation/methods*

The probiotic strain Escherichia coli Nissle 1917 (EcN) with high biocompatibility and susceptibility to genetic modification is often applied in bacterial therapies for cancer. However, most studies have used plasmids as vectors to construct engineering strains from EcN. Plasmid-based expression systems suffer from genetic instability, and they need antibiotic selective pressure to maintain high copy number. This study aimed to employ EcN for synthesizing the photosensitizer 5-aminolevulinic acid (5-ALA). Firstly, the key genes of 5-ALA synthesis, hemA^M and hemL, were integrated into the EcN genome by the phage integration technique. Then, chemically inducible chromosomal evolution (CIChE) was adopted to increase the copy number of hemA^M and hemL and thus improved the stable synthesis of 5-ALA. The in vitro cell experiments verified that the constructed engineering strain can deliver stably synthesized 5-ALA to tumor cells and inhibit their growth. This study provided a basis for applying the engineering strains of EcN in the photodynamic therapy for tumors.
Escherichia coli/metabolism* ; Aminolevulinic Acid/metabolism* ; Photosensitizing Agents/pharmacology* ; Plasmids/genetics* ; Chromosomes, Bacterial/genetics* ; Genetic Engineering ; Humans ; Probiotics ; Photochemotherapy

Although 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) has been demonstrated to be a novel and effective therapeutic modality for some human malignancies, its effect and mechanism on glioma are still controversial. Previous studies have reported that 5-ALA-PDT induced necrosis of C6 rat glioma cells in vitro. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on C6 gliomas implanted in rats in vivo. Twenty-four rats bearing similar size of subcutaneously implanted C6 rat glioma were randomly divided into 3 groups: receiving 5-ALA-PDT (group A), laser irradiation (group B), and mock procedures but without any treatment (group C), respectively. The growth, histology, microvessel density (MVD), and apoptosis of the grafts in each group were determined after the treatments. As compared with groups B and C, the volume of tumor grafts was significantly reduced (P<0.05), MVD was significantly decreased (P<0.001), and the cellular necrosis was obviously increased in group A. There was no significant difference in apoptosis among the three groups. The in vivo studies confirmed that 5-ALA-PDT may be an effective treatment for gliomas by inhibiting the tumor growth. The mechanism underlying may involve increasing the cellular necrosis but not inducing the cellular apoptosis, which may result from the destruction of the tumor microvessels.
Aminolevulinic Acid ; pharmacology ; therapeutic use ; Animals ; Brain Neoplasms ; blood supply ; drug therapy ; pathology ; Cell Line, Tumor ; Glioma ; blood supply ; drug therapy ; pathology ; Microvessels ; drug effects ; Photochemotherapy ; Photosensitizing Agents ; pharmacology ; therapeutic use ; Rats ; Rats, Wistar ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effect of 8-aminolevulinic acid (ALA) photodynamic therapy (PDT) on the cell proliferation, apoptosis and collagen secretion in keloid fibroblasts and to provide the theoretical base for ALA-PDT treatment of keloids.

METHODSFibroblasts from keloid patients were cultured to the third generation in vitro and incubated in 0, 1, 3, 6, 9 mmol/L of δ-aminolevulinic acid for 3 h in the darkness. Then they were exposed to 635 nm wavelength red light ( 30 J/cm2 ) and continued incubation 24 h after irradiation. CCK-8 assay was used to detect proliferation inhibition rate of fibroblasts. The content of hydroxyproline was measured by colorimetric method. The expression of p-Akt and programmed cell death 4 ( PDCD4) were detected by Western blot.

RESULTSThe inhibition rate of keloid fibroblasts were respectively 0, (8.30 ± 1.01)%, (29.48 ± 3.27)%, (52.01 ± 5.34)%, (79.99 ± 5.85)% with the presence of difference concentrations (0, 1, 3, 6, 9 mmol/L) of ALA. The content of hydroxyproline were respectively (9.540 0 ± 0.352 42), (6.242 5 ± 0.224 85 ), (5.107 5 ± 0.534 88), (3.490 0 ± 0.623 48), (2.945 0 ± 0.514 10) μg/mg. The relative expression of p-Akt were respectively 1, 0.75 ± 0.12, 0.52 ± 0.14, 0.41 ± 0.18, 0.32 ± 0.09. The relative expression of PDCD4 were respectively 1, 1.18 ± 0.19, 1.51 ± 0.22, 0.15 ± 0.30, 2.44 ± 0.22. The difference was statistically significant when compared the group of 1, 3, 6, 9 mmol/L with 0 mmol/L (P < 0.05).

CONCLUSIONSIn concentration within the range of 1-9 mmol/L, ALA could inhibit the proliferation of fibroblasts significantly, promote fibroblasts apoptosis and reduce the content of hydroxyproline in a dose-dependent manner, indicating that 8-aminolevulinic acid photodynamic therapy may be a potential treatment for keloid.

Aminolevulinic Acid ; pharmacology ; Apoptosis ; drug effects ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Collagen ; secretion ; Fibroblasts ; cytology ; drug effects ; secretion ; Humans ; Keloid ; drug therapy ; pathology ; Light ; Photochemotherapy ; methods ; Photosensitizing Agents ; pharmacology

This study was purposed to investigate the changes of mitochondrial membrane potential (MMP) and apoptosis-related gene Bcl-2 expression of HL-60 cells treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). HL-60 cell line was used as a model and divided into 4 groups: ALA group, PDT group, ALA+PDT group and control group. The change of MMP was detected by flow cytometry with JC-1 (lipophilic cation 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-benzimidazol-carbocyanine iodide); the mRNA expression of Bcl-2 was determined by semi-quantitative RT-PCR and real-time PCR. The results demonstrated that MMP significantly decreased after treatment with ALA-PDT and the ratio of cells with disrupted MMP obviously increased in ALA+PDT group in time-dependence manner, as compared with control, ALA and PDT groups (P < 0.05), while no difference between ALA and PDT groups was found. The semi-quantitative RT-PCR and real-time PCR showed that the expression level of Bcl-2 was obviously down regulated at 2 h after ALA-PCT, further down-regulated at 4 h, and lasted in low level at 24 h. It is concluded that ALA-PDT-induced apoptosis of HL-60 cells is associated with its effect on MMP, that is ALA-PDT promotes cell apoptosis through effect on mitochondrial function.
Aminolevulinic Acid ; pharmacology ; Apoptosis ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Photochemotherapy ; Photosensitizing Agents ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo find a method for improving the salt resistance of seeds and seedlings for Perilla Frutescens under NaCl stress, seed germination and physiological characteristics of P. frutescens seedlings were studied.

METHODSeveral physiological indexes of P. frutescens seeds treated with different concentrations of Ca2+, 5-aminolevulinic acid (ALA), salicylic acid (SA) and spermidine (Spd) under NaCl stress like the germination vigor, germination rate, germination index and vigor index were measured. And other indexes like the biomass of the seedlings, the content of malondialdehyde (MDA) in leaves, the activities of superoxide (SOD), peroxidase (POD) and catalase (CAT) were also measured.

RESULTThe germination of P. frutescens seeds under NaCl stress (100 mmol x L(-1)) was inhibited obviously. But after the treatment with Ca2+, ALA , SA and Spd, all germination indexes were increased. Ca2+ (10 mmol x L(-1)), ALA (100 mg x L(-1)), SA (50 mg x L(-1)) and Spd (0.25 mmol x L(-1)) could obviously alleviate the damage of salt stress to the seeds of P. frutescens. ALA (100 mg x L(-1)) significantly increased all indexes. The germination vigor was 65.3%, the germination rate was 89.7%, the germination index and vigor index were 15.2 and 0.1238, respectively. All treatments decreased the content of MDA in leaves. The activities of three enzymes including SOD, POD and CAT were all increased. ALA (100 mg x L(-1)) had the enzymes activity reach the maximum with 0.72, 6, 82 and 5.64 U x mg(-1), respectively.

CONCLUSIONCa2+ ALA , SA and Spd with appropriate concentration could significantly alleviate the damages to the seeds and seedlings of P. frutescens under NaCl stress and promote the salt resistance of the seeds and seedlings.

Aminolevulinic Acid ; pharmacology ; Calcium ; pharmacology ; Catalase ; metabolism ; Dose-Response Relationship, Drug ; Germination ; drug effects ; Malondialdehyde ; metabolism ; Perilla frutescens ; drug effects ; enzymology ; metabolism ; physiology ; Peroxidase ; metabolism ; Salicylic Acid ; pharmacology ; Seedlings ; drug effects ; enzymology ; metabolism ; physiology ; Sodium Chloride ; pharmacology ; Spermidine ; pharmacology ; Stress, Physiological ; drug effects ; Superoxide Dismutase ; metabolism

Adolescent ; Aminolevulinic Acid ; pharmacology ; Female ; Humans ; Porphyria, Acute Intermittent ; physiopathology

Aminolevulinic acid (ALA) is biosynthesized by the enzyme ALA synthase (ALAS). The ALA production has been studied using the overproducing ALAS from several bacteria in Escherchia coil, respectively. However, ALAS from eucaryote expressed in E. coli for producing ALA in the culture is not known. The extracellular ALA production and cell growth were investageted respectively using the recombinant E. coli overproducing Saccharomyces cerevisiae ALAS in shake-flask fermentations. The ALAS activity from the cell extract was assayed. The extracellular ALA was purified by the national-made large-dimension resins and confirmed by the capillary electrophoresis measurements. At 12h after induction at 37 degrees C, the extracellular ALA production was up to 162mg per liter LB culture at initial pH 6.5 with exogenous levulinate, succinate and and glycine at the concentration of 20 mmol/L respectively. The purity of ALA after purification is up to 90%.
5-Aminolevulinate Synthetase ; genetics ; metabolism ; Aminolevulinic Acid ; isolation & purification ; metabolism ; Cell Division ; drug effects ; Dose-Response Relationship, Drug ; Electrophoresis, Capillary ; Escherichia coli ; genetics ; growth & development ; metabolism ; Extracellular Space ; drug effects ; metabolism ; Glycine ; pharmacology ; Hydrogen-Ion Concentration ; Levulinic Acids ; pharmacology ; Recombinant Proteins ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Succinic Acid ; pharmacology

This experiment was designed to explore the pattern of K562 and HL60 leukemia cells death, the effects on their cell cycle and the cytoplasmic free calcium concentration ([Ca2+]i) induced by 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Under the transmission electron microscope (TEM), two kinds of leukemia cells' ultrastructure were observed. Flow cytometry combined with Annexin V-FITC/PI labeling was used to detect the pattern of K562 and HL60 cells' death induced by ALA-PDT. Flow cytometry combined with PI labeling was used to analyze the change in the cell cycle induced by ALA-PDT, and confocal laser scanning microscopy (CLSM) combining with calcium fluorescence probe was used to detect the change in the cytoplasmic free calcium concentration ([Ca2+]i). Immediately after irradiation, many typical apoptotic bodies were seen in the cells treated. Most of the cells treated were necrotic at 24 hours following irradiation. Flow cytometry analysis suggested that the main patterns of the cells' death were apoptosis immediately after irradiation and necrosis post-apoptosis at 24 hours post irradiation. Immediately and 24 hours after irradiation, the proportion of S phase of K562 was 57. 67% +/- 1.13% and 84.77% +/- 6.20% respectively, and the proportion of S phase of HL60 was 74.60% +/- 7.27% and 84.60% 1.74% respectively. Both [Ca+]i of the treated K562 and HL60 were increased obviously. In the best experiment condition, the initial pattern of the K562 and HL60 leukemia cells' death induced by PDT was apoptosis and the main pattern was necrosis post apoptosis. The two kinds of cells were arrested at S phase by ALA-PDT. During the death of the leukemia cells, the increase in intracellular free calcium concentration could be responsible for the ALA photodynamically induced damage to K562 and HL60 cells.
Aminolevulinic Acid ; pharmacology ; Apoptosis ; drug effects ; Calcium ; chemistry ; Cell Division ; drug effects ; Cytosol ; chemistry ; HL-60 Cells ; drug effects ; Humans ; K562 Cells ; drug effects ; Photochemotherapy

BACKGROUNDWe assessed whether the CaNa2 EDTA could improve the accumulation of protoporphyrin IX (PpIX) and photosensitisation in HEp-2 cells as well as the depth of treatment of skin cancers on the topical 5-Aminolaevulinic acid (5-ALA) PDT.

METHODSHEp-2 cells were incubated with 5-ALA (0-1 mmol/L) and CaNa2EDTA (0-1 mmol/L) for 4 hours, intracellular protoporphyrin IX content was quantified by extraction, and cell viability was assessed by use of the methyl-tetrazolium (MTT) assay four hours after exposure to light. In comparison with the pictures before and after treatment, depth of treatment could be determined using a Acuson Sequioa 512 phase-array system in paired experiments.

RESULTSPpIX accumulation increased with increasing extracellular concentrations of ALA (0-1 mmol/L). Adding 1 mmol/L of CaNa2EDTA increased 30% PpIX accumulation over the same period of incubation in the concentration of 1 mmol/L ALA. Significant difference was observed between the 5-ALA alone group and 5-ALA combined CaNa2 EDTA group in the PpIX accumulation (P < 0.01). Cell viability after exposure to light decreased with adding CaNa2 EDTA, a statistical difference in a same fluence above 1.2 J/cm2 between two groups was demonstrated (P < 0.05, P < 0.01 respectively). Depth of treatment of skin cancers were increased in CaNa2 EDTA-treated group.

CONCLUSIONCaNa2 EDTA could improve the PpIX accumulation and photosensitisation in HEp-2 cells. Clinically, CaNa2 EDTA could increase the depth of treatment in the cutaneous cancers.

Aminolevulinic Acid ; therapeutic use ; Cell Line, Tumor ; Cell Survival ; Edetic Acid ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Photochemotherapy ; Protoporphyrins ; analysis ; Skin Neoplasms ; drug therapy