In vitro evaluation of the efficacy of photodynamic therapy using 5-ALA on homologous feline mammary tumors in 2D and 3D culture conditions and a mouse subcutaneous model with 3D cultured cells.

BACKGROUND: Numerous studies have shown that photodynamic therapy (PDT) has a therapeutic effect on mammary tumor cells, with 5-aminolevulinic acid (5-ALA-HCL) being a commonly used photosensitizer for PDT. Feline mammary tumors (FMTs) are relatively common. However, the cytotoxic and antitumor effects of 5-ALA-PDT on FMTs have not been clarified. To this end, we evaluated the therapeutic effect of 5-ALA-PDT on FMTs through in vitro experiments using an FMT FKR cell line established for this study. METHODS: We performed 5-ALA-PDT in 2D-cultured FKR-A (adherent cells) and 3D-cultured FKR-S (spheroid cells) cells and performed a series of studies to evaluate the cell viability and determine the protoporphyrin IX (PpIX) content in the cells as well as the expression levels of mRNAs associated with PpIX production and release. An in vivo study was performed to assess the effectiveness of 5-ALA-PDT. RESULTS: There was a significant difference in the concentration of PpIX in FMT cells under different incubation culture modes (2D versus 3D culture). The concentration of PpIX in FMT cells was correlated with the differences in cell culture (2D and 3D) as well as the expression levels of genes such as PEPT1, PEPT2, FECH, and HO-1. CONCLUSIONS: In the in vitro study, 5-ALA-PDT had a stronger inhibitory effect on 3D-cultured FKR-S cells, which resemble the internal environment of organisms more closely. We also observed a significant inhibitory effect of 5-ALA-PDT on FMT cells in vivo. To our knowledge, this is the first study on 5-ALA-PDT for FMTs under both 2D and 3D conditions.

Mechanism of Differential Susceptibility of Two (Canine Lung Adenocarcinoma) Cell Lines to 5-Aminolevulinic Acid-Mediated Photodynamic Therapy.

Photodynamic therapy (PDT) is a clinically approved, minimally invasive treatment for malignant tumors. Protoporphyrin IX (PpIX), derived from 5-aminolevulinic acid (5-ALA) as the prodrug, is one of the photosensitizers used in PDT. Recently, we reported a significant difference in response to 5-ALA-mediated PDT treatment in two canine primary lung adenocarcinoma cell lines (sensitive to PDT: HDC cells, resistant to PDT: LuBi cells). This study aimed to examine the difference in cytotoxicity of 5-ALA-mediated PDT in these cells. Although intracellular PpIX levels before irradiation were similar between HDC and LuBi cells, the percentage of ROS-positive cells and apoptotic cells in LuBi cells treated with 5-ALA-mediated PDT was significantly lower than that in HDC cells treated with 5-ALA-mediated PDT. A high dosage of the NO donor, DETA NONOate, significantly increased the cytotoxicity of 5-ALA-mediated PDT against LuBi cells. These results suggest that the sensitivity of 5-ALA-mediated PDT might be correlated with NO.

The sustaining of fluorescence in photodynamic diagnosis after the administration of 5-aminolevulinic acid in carcinogen-induced bladder cancer orthotopic rat model and urothelial cancer cell lines.

BACKGROUND: The administration of 5-aminolevulic acid hydrochloride (5-ALA·HCl) 3 h (range: 2-4 h) before photodynamic diagnosis (PDD) is recommended for detecting bladder tumors. However, there is insufficient evidence on the time duration for the fluorescence of PDD after oral administration of 5-ALA. We investigated the sustainability of the photodynamic effect and protoporphyrinⅨ (PpⅨ) after 5-ALA administration in a carcinogen-induced bladder tumor rat model and bladder cancer cell lines. METHODS: The carcinogen-induced bladder tumor orthotopic rat model was established by the administration of N-butyl-N-(4-hydroxybutyl) nitrosamine. RESULTS: Red fluorescence was visible 2-8 h after the oral administration of 5-ALA in the carcinogen-induced bladder tumor rat model. Plasma and intratissue PpⅨ (nM) progressed to a higher level at 2 h and remained almost constant 2-8 h after oral administration of 5-ALA. The peak fluorescence intensity of PpⅨ was observed 3-4 h after the administration of 5-ALA in bladder cancer cell lines. The accumulated PpⅨ remained for 4 h after the removal of 5-ALA in UMUC3 cells. It was not clearly visible 3 h after the removal of 5-ALA in MGHU3 and T24 cells. The expression level of ferrochelatase was significantly lower in UMUC3 cells than in other cells. Our findings suggest that 5-ALA-assisted PDD (ALA-PDD) can aid in detecting non-muscle-invasive bladder cancer 2-8 h after 5-ALA administration. CONCLUSION: Urologists might not be required to make excess effort to start ALA-PDD-assisted transurethral resection of bladder tumor after the administration of 5-ALA.

Effectiveness of Photodynamic Screening Using 5-Aminolevulinic Acid for the Diagnosis of Pancreatic Cancer.

BACKGROUND/AIM: We evaluated urinary levels of porphyrin metabolites, such as uroporphyrin (UP) and coproporphyrin (CP), after 5-Aminolevulinic acid (ALA) administration in patients with or without pancreatic cancer (PaC). PATIENTS AND METHODS: Sixty-seven subjects with PaC, 11 with pancreatitis, and 9 with normal pancreas (NP) were enrolled. Urine samples from all subjects were collected prior to ALA administration and at more than 4 hours after ALA administration. We measured the urinary levels of UP and CP by high-performance liquid chromatography analysis. RESULTS: The PaC group showed significantly higher UP levels compared to NP groups (104.9 nmol/g Cre vs. 53.4 nmol/g Cre, p=0.014). Moreover, PaC patients with long-term survival had significantly lower urinary levels of UP at diagnosis (98.8 nmol/gCre) than the short-term survival group (125.2 nmol/gCre) (p=0.042). CONCLUSION: The urinary levels of UP after ALA administration might serve as a promising biomarker for diagnosis and prognosis prediction of PaC.

Efficacy of 5-Aminolevulinic Acid in Photodynamic Detection and Photodynamic Therapy in Veterinary Medicine.

5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA in various veterinary tumors and treated canine and feline tumors. 5-ALA-PDD sensitivity and specificity in the whole sample group for dogs and cats combined were 89.5 and 50%, respectively. Notably, some small tumors disappeared upon 5-ALA-PDT. Although single PDT application was not curative, repeated PDT+/-chemotherapy achieved long-term tumor control. We analyzed the relationship between intracellular PpIX concentration and 5-ALA-PDT in vitro cytotoxicity using various primary tumor cells and determined the correlation between intracellular PpIX concentration and 5-ALA transporter and metabolic enzyme mRNA expression levels. 5-ALA-PDT cytotoxicity in vitro correlated with intracellular PpIX concentration in carcinomas. Ferrochelatase mRNA expression levels strongly negatively correlated with PpIX accumulation, representing the first report of a correlation between mRNA expression related to PpIX accumulation and PpIX concentration in canine tumor cells. Our findings suggested that the results of 5-ALA-PDD might be predictive for 5-ALA-PDT therapeutic effects for carcinomas, with 5-ALA-PDT plus chemotherapy potentially increasing the probability of tumor control in veterinary medicine.

5-Aminolevulinic acid with ferrous iron improves early renal damage and hepatic steatosis in high fat diet-induced obese mice.

5-Aminolevulinic acid, a natural amino acid, activates mitochondrial respiration and induces heme oxygenase-1 expression. Obesity and type 2 diabetes mellitus are associated with age-related mitochondrial respiration defect, oxidative stress and inflammation. The aim of this study is to investigate the effects of 5-aminolevulinic acid with sodium ferrous citrate on early renal damage and hepatic steatosis. 7-Month-old C57BL/6 mice were fed with a standard diet or high fat diet for 9 weeks, which were orally administered 300 mg/kg 5-aminolevulinic acid combined with 47 mg/kg sodium ferrous citrate (5-aminolevulinic acid/sodium ferrous citrate) or vehicle for the last 5 weeks. We observed that 5-aminolevulinic acid/sodium ferrous citrate significantly decreased body weight, fat weight, hepatic lipid deposits and improved levels of blood glucose and oral glucose tolerance test. In addition, 5-aminolevulinic acid/sodium ferrous citrate suppressed increased glomerular tuft area in high fat diet-fed mice, which was associated with increased heme oxygenase-1 protein expression. Our findings demonstrate additional evidence that 5-aminolevulinic acid/sodium ferrous citrate could improve glucose and lipid metabolism in diabetic mice. 5-Aminolevulinic acid/sodium ferrous citrate has potential application in obesity or type 2 diabetes mellitus-associated disease such as diabetic nephropathy and nonalcoholic fatty liver disease.

Dynamics of absorption, metabolism, and excretion of 5-aminolevulinic acid in human intestinal Caco-2 cells.

5-Aminolevulinic acid (ALA) is a precursor for the biosynthesis of porphyrins and heme. Although the oral administration of ALA has been widely applied in clinical settings, the dynamics of its absorption, metabolism, and excretion within enterocytes remain unknown. In this study, after enterocytic differentiation, Caco-2 cells were incubated with 200 µM ALA and/or 100 µM sodium ferrous citrate (SFC) for up to 72 h. Both ALA and the combination of ALA and SFC promoted the synthesis of heme, without affecting the expression of genes involved in intestinal iron transport, such as DMT1 and FPN. The enhanced heme synthesis in Caco-2 cells was more pronounced under the effect of the combination of ALA and SFC than under the effect of ALA alone, as reflected by the induced expression of heme oxygenase 1 (HO-1), as well as a reduced protein level of the transcriptional corepressor Bach1. Chromatin immunoprecipitation analysis confirmed Bach1 chromatin occupancy at the enhancer regions of HO-1, which were significantly decreased by the addition of ALA and SFC. Finally, Transwell culture of Caco-2 cells suggested that the administered ALA to the intestinal lumen was partially transported into vasolateral space. These findings enhance our understanding of the absorption and metabolism of ALA in enterocytes, which could aid in the development of a treatment strategy for various conditions such as anemia.

5-aminolevulinic acid combined with ferrous ion reduces adiposity and improves glucose tolerance in diet-induced obese mice via enhancing mitochondrial function.

BACKGROUND: Mitochondrial dysfunction is associated with obesity and various obesity-associated pathological conditions including glucose intolerance. 5-Aminolevulinic acid (ALA), a precursor of heme metabolites, is a natural amino acid synthesized in the mitochondria, and various types of cytochromes containing heme contribute to aerobic energy metabolism. Thus, ALA might have beneficial effects on the reduction of adiposity and improvement of glucose tolerance through its promotion of heme synthesis. In the present study, we investigated the effects of ALA combined with sodium ferrous citrate (SFC) on obesity and glucose intolerance in diet-induced obese mice. METHODS: We used 20-weeks-old male C57BL/6J diet-induced obesity (DIO) mice that had been fed high-fat diet from 4th week or wild-type C57BL/6J mice. The DIO mice were orally administered ALA combined with SFC (ALA/SFC) for 6 weeks. At the 4th and 5th week during ALA/SFC administration, mice were fasted for 5 h and overnight, respectively and used for oral glucose tolerance test. After the ALA/SFC administration, the plasma glucose levels, weight of white adipose tissue, and expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes were examined. Furthermore, the effects of ALA/SFC on lipid content and glucose uptake were examined in vitro. RESULTS: Oral administration of ALA/SFC for 6 weeks reduced the body weight by about 10% and the weight of white adipose tissues in these animals. In vitro, ALA/SFC reduced lipid content in mouse 3T3-L1 adipocytes in a dose dependent manner, and enhanced glucose uptake in 3T3-L1 adipocytes by 70-90% and rat L6 myoblasts by 30% at 6 h. Additionally, oral administration of ALA/SFC reduced plasma glucose levels and improved glucose tolerance in DIO mice. Furthermore, ALA/SFC enhanced the expression of OXPHOS complexes III, IV, and V by 40-70% in white adipose tissues of DIO mice, improving mitochondrial function. CONCLUSIONS: Our findings indicate that ALA/SFC is effective in the reduction of adiposity and improvement of glucose tolerance, and that the induction of mitochondrial OXPHOS complex III, IV, and V by ALA/SFC might be an essential component of the molecular mechanisms underlying these effects. ALA/SFC might be a useful supplement for obesity and obesity-related metabolic disease such as type 2 diabetes mellitus.

Combination of 5-aminolevulinic acid and ferrous ion reduces plasma glucose and hemoglobin A1c levels in Zucker diabetic fatty rats.

Mitochondrial dysfunction is associated with type 2 diabetes mellitus (T2DM). 5-Aminolevulinic acid (ALA), a natural amino acid produced only in the mitochondria, is a precursor of heme. Cytochromes that contain heme play an important role in aerobic energy metabolism. Thus, ALA may help reduce T2DM-associated hyperglycemia. In this study, we investigated the effect of ALA combined with sodium ferrous citrate (SFC) on hyperglycemia in Zucker diabetic fatty (ZDF) rats. We found that the gavage administration of ALA combined with SFC (ALA/SFC) for 6 weeks reduced plasma glucose and hemoglobin A1c (HbA1c) levels in rats without affecting plasma insulin levels. The glucose-lowering effect depended on the amount of ALA/SFC administered per day. Furthermore, the glucose tolerance was also significantly improved by ALA/SFC administration. Although food intake was slightly reduced in the rats administered ALA/SFC, there was no effect on their body weight. Importantly, ALA/SFC administration induced heme oxygenase-1 (HO-1) expression in white adipose tissue and liver, and the induced expression levels of HO-1 correlated with the glucose-lowering effects of ALA/SFC. Taken together, these results suggest that ALA combined with ferrous ion is effective in reducing hyperglycemia of T2DM without affecting plasma insulin levels. HO-1 induction may be involved in the mechanisms underlying the glucose-lowering effect of ALA/SFC.

Urinary 5-Aminolevulinic Acid Concentrations as a Potential Tumor Marker for Colorectal Cancer Screening and Recurrence.

BACKGROUND/AIM: Tumor biomarkers, such as carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9), are used to screen and monitor tumor recurrence in patients with colorectal cancer (CRC). 5-Aminolevulinic acid (5-ALA) is used in photodynamic diagnosis and therapy. Porphyrins produced by tumor cells are excreted in the urine after 5-ALA administration. In this study, we evaluated the use of porphyrins as novel tumor markers in urine samples from patients with CRC. PATIENTS AND METHODS: Porphyrin metabolite concentrations were measured in urine samples of 33 patients with CRC, 16 patients with benign disease and 8 healthy adults, after 5-ALA administration. RESULTS: The porphyrin metabolite concentrations were significantly increased in the CRC group compared to the control group, while in CRC patients, the porphyrin metabolite concentrations in urine were significantly decreased after surgery. CONCLUSION: These results suggest that the measurement of porphyrin metabolites in urine may potentially serve as a new screening and recurrence marker for CRC.

Prolonged Mouse Cardiac Graft Cold Storage via Attenuating Ischemia-Reperfusion Injury Using a New Antioxidant-Based Preservation Solution.

BACKGROUND: One of the major events in ischemia-reperfusion (I/R)-induced heart injury in cardiac transplantation is the generation of reactive oxygen species. We hypothesized that a novel preservation solution called SBI-SEIIKU II (SS-II) contains 3 antioxidant reagents: L-cysteine, glycine, ascorbic acid/ascorbic acid-2-phosphate magnesium, which can block the generation of reactive oxygen species to result in a prolongation of the cold storage time via attenuating I/R injury. METHODS: C57BL/6CrSlc(B6) mice underwent syngeneic mice heterotopic heart transplantation, and the animals were derived into 3 groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in histidine-tryptophan-ketoglutarate (HTK) for 24 and 48 hours (HTK group), and recipients with grafts preserved in SS-II for 24 and 48 hours (SS-II group). RESULTS: After 48 hours of preservation, there were no grafts that survived in the HTK group; however, the SS-II group had a high survival rate. After 24 hours of preservation, SS-II decreased the oxidative damage, myocardial apoptosis, and the infiltration of macrophages and neutrophils in the cardiac grafts in the early phase and suppressed the development of myocardial fibrosis in long-term grafts compared with HTK. CONCLUSIONS: The SS-II prolongs the acceptable cold storage time and protects the myocardium from I/R injury via inhibiting oxidative stress-associated damage. We believe that this novel preservation solution may be simple and safe for use in the clinical transplantation field.

Plasma protoporphyrin IX following administration of 5-aminolevulinic acid as a potential tumor marker.

Exogenously administered 5-aminolevulinic acid (ALA) is metabolized to protoporphyrin IX (PpIX), which specifically accumulates in cancer cells and emits red fluorescence by blue light irradiation. These phenomena are applied for the intraoperative diagnosis of cancer. Based on the fact that accumulated PpIX in cancer cells is exported extracellularly via the ATP-binding cassette transporter G2, we hypothesized that the measurement of plasma PpIX concentrations could be applied as a tumor marker for cancer screening. In the present study, the use of plasma samples from bladder cancer patients were evaluated as a tumor marker. ALA, 1.0 g, was orally administered to bladder cancer patients and healthy adults. The plasma concentration of PpIX was measured using a high-performance liquid chromatography system. The plasma PpIX concentration following ALA administration was significantly higher in bladder cancer patients than that in the healthy adults, suggesting the effectiveness of plasma PpIX analysis following ALA administration for cancer screening. Additionally, 4 h after ALA administration, plasma PpIX showed high sensitivity (94.4%) and high specificity (80.0%).

Porphyrins as urinary biomarkers for bladder cancer after 5-aminolevulinic acid (ALA) administration: the potential of photodynamic screening for tumors.

BACKGROUND: Tumor biomarkers are commonly used for cancer screening and as indicators of treatment effects. We recently reported that urine porphyrin levels from tumor-bearing mice were elevated compared with those from normal mice after administration of 5-aminolevulinic acid (ALA). In the present study, we evaluated the use of urine samples from bladder cancer patients as tumor biomarkers. METHODS: ALA, 1.0 g, was orally administered to 66 bladder cancer patients and 20 healthy adults. The urine concentrations of uroporphyrin I (UPI), uroporphyrin III (UPIII), coproporphyrin I (CPI), coproporphyrin III (CPIII), and total porphyrins were measured using High Performance Liquid Chromatography (HPLC) system. RESULTS: Almost all of the urinary porphyrin concentrations from the patients with bladder cancer were higher than those from healthy adults. Moreover, 8h after ALA administration, urinary UPI and CPI showed high sensitivity (100 for UPI and CPI) and specificity (96.4 for UPI and 91.4 for CPI). CONCLUSION: These results indicate that the presence of urinary porphyrins after administration of ALA may function as tumor biomarkers. This method represents a possible new tumor screening method called photodynamic screening (PDS) using ALA-induced porphyrins.