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.

5-Aminolevulinic acid bypasses mitochondrial complex I deficiency and corrects physiological dysfunctions in Drosophila.

Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.

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.

5-Aminolevulinic acid/sodium ferrous citrate enhanced the antitumor effects of programmed cell death-ligand 1 blockade by regulation of exhausted T cell metabolism in a melanoma model.

Mitochondria are key cytoplasmic organelles. Their activation is critical for the generation of T cell proliferation and cytotoxicity. Exhausted tumor-infiltrating T cells show a decreased mitochondrial function and mass. 5-Aminolevulinic acid (5-ALA), a natural amino acid that is only produced in the mitochondria, has been shown to influence metabolic functions. We hypothesized that 5-ALA with sodium ferrous citrate (SFC) might provide metabolic support for tumor-infiltrating T cells. In a mouse melanoma model, we found that 5-ALA/SFC with a programmed cell death-ligand 1 (PD-L1) blocking Ab synergized tumor regression. After treatment with 5-ALA/SFC and anti-PD-L1 Ab, tumor infiltrating lymphocytes (TILs) were not only competent for the production of cytolytic particles and cytokines (granzyme B, interleukin-2, and γ-interferon) but also showed enhanced Ki-67 activity (a proliferation marker). The number of activated T cells (PD-1+ Tim-3- ) was also significantly increased. Furthermore, we found that 5-ALA/SFC activated the mitochondrial functions, including the oxygen consumption rate, ATP level, and complex V expression. The mRNA levels of Nrf-2, HO-1, Sirt-1, and PGC-1α and the protein levels of Sirt-1 were upregulated by treatment with 5-ALA/SFC. Taken together, our findings revealed that 5-ALA/SFC could be a key metabolic regulator in exhausted T cell metabolism and suggested that 5-ALA/SFC might synergize with anti-PD-1/PD-L1 therapy to boost the intratumoral efficacy of tumor-specific T cells. Our study not only revealed a new aspect of immune metabolism, but also paved the way to develop a strategy for combined anti-PD-1/PD-L1 cancer immunotherapy.

Effects of 5-aminolevulinic acid with iron supplementation on respiratory responses to graded cycling and interval walking training achievement in older women over 75 yrs.

BACKGROUND: Exercise training above a given intensity is necessary to prevent age-associated physical disability and diseases; however, the physical and psychological barriers posed by deteriorated physical fitness due to aging may hinder older people from performing daily exercise training. Because 5-aminolevulinic acid (ALA), a precursor of heme, reportedly improves mitochondrial function, we examined whether ALA, combined with sodium ferrous citrate (SFC) for enhancement, improved aerobic capacity and voluntary exercise training achievement in older women aged over 75 yrs. METHODS: The study was conducted using a placebo-controlled, double-blind crossover design. Fifteen women aged ~78 yrs. with no exercise habits underwent two trials for 7 days each where they performed interval walking training (IWT), repeating fast and slow speeds of walking for 3 min each, at >70% and at ~40% of peak aerobic capacity for walking, respectively, with ALA+SFC (100 and 115 mg/day, respectively) or placebo supplement intake (CNT), with a 12-day washout period. Before and after each trial, subjects underwent a graded cycling test while having their oxygen consumption rate (V·O2), carbon dioxide production rate (V·CO2), and plasma lactate concentration ([Lac-]p) measured. Furthermore, during the supplement intake period, exercise intensity for IWT was measured by accelerometry. RESULTS: In ALA+SFC, the increases in V·O2 and V·CO2 during the graded cycling test were attenuated (both, P < 0.01) with a 13% reduction in [Lac-]p (P = 0.012) while none of these attenuated responses occurred in CNT (all, P > 0.46). Furthermore, energy expenditure and time during fast walking for IWT were 25% (P = 0.032) and 21% (P = 0.022) higher in ALA+SFC than in CNT. CONCLUSION: Thus, ALA+SFC supplementation improved aerobic capacity and thus increased fast-walking training achievement in older women.

Efficiency of aminolevulinic acid (ALA)-photodynamic therapy based on ALA uptake transporters in a cell density-dependent malignancy model.

The effectiveness of the conventional chemotherapy for cancer are compromised as the cancer cells advances in their malignancy level as they acquired drug resistance. In this study, we aimed to evaluate the efficiency of aminolevulinic acid-photodynamic therapy (ALA-PDT) against cancer of various malignancy levels, indicated by the expression level of receptor associated nuclear factor-κB ligand (RANKL), through the expression levels of ALA uptake transporters. We established a malignancy model by gradually increasing the cell density of cancer cells. Western blotting was used to study the expression levels of RANKL, ALA uptake transporters and the cell density-dependent Yes-associated protein (YAP) under different cell densities. The amount of protoporphyrin (PpIX) produced and cell viability were then studied using high performance liquid chromatography (HPLC) and ALA-PDT assay. Our study showed that the amount of PpIX production doubled in high cell density/cancer malignancy cultures and the effectiveness of ALA-PDT when subjected to light irradiation at 635 nm are significantly at higher cancer malignancy. We observed that the expression levels of ALA uptake transporters and YAP correlated with higher cell density/cancer malignancy, suggesting a possible relationship among these three factors. These findings suggest that ALA-PDT is more effective in cancer cells of higher malignancy due to the upregulation of transporters involved in ALA uptake.

5-Aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) ameliorated liver injury in a murine acute graft-versus-host disease model by reducing inflammation responses through PGC1-α activation.

Acute graft-versus-host disease (aGvHD) remains lethal as a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). Inflammatory responses play an important role in aGvHD. 5-Aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) has been widely reported to have a major effect on the anti-inflammatory response; however, these effects in aGvHD models have never been reported. In this study, a murine aGvHD model was developed by transferring spleen cells from donor B6/N (H-2kb) mice into recipient B6D2F1 (H-2kb/d) mice. In addition to evaluating manifestations in aGvHD mice, we analyzed the serum ALT/AST levels, liver pathological changes, infiltrating cells and mRNA expression of inflammation-related cytokines and chemokines. 5-ALA/SFC treatment significantly ameliorated liver injury due to aGvHD and decreased the population of liver-infiltrating T cells, resulting in a reduced expression of pro-inflammatory cytokines and chemokines. Furthermore, the mRNA expression proliferator-activated receptor-γcoactivator (PGC-1α) was enhanced, which might explain why 5-ALA/SFC treatment downregulates inflammatory signaling pathways. Our results indicated that 5-ALA/SFC can ameliorate liver injury induced by aGvHD through the activation of PGC-1α and modulation of the liver mRNA expression of inflammatory-related cytokines and chemokines. This may be a novel strategy for treating this disease.

Efficacy of 5-aminolevulinic acid and LED photodynamic therapy in cervical intraepithelial neoplasia: A clinical trial.

BACKGROUND: 5-Aminolaevulinic acid (5ALA) is a precursor of the strong sensitizer protoporphyrin IX (PpIX) in the heme synthesis pathway. We conducted aclinical trial designed to evaluate the efficacy and safety of 5ALA photodynamic therapy (PDT) using a light-emitting diode (LED) in patients with cervical intraepithelial neoplasia (CIN). METHODS: Data for 51 CIN patients who underwent 5ALA-PDT between 2012 and 2017 were prospectively analysed. After a 20 % 5ALA jelly formulation was topically applied to the cervix, the region was irradiated with red light at approximately 633 nm to excite PpIX for treatment. We estimated outcomes by cytology, pathology, and human papilloma virus (HPV) testing after PDT. RESULTS: Patients underwent two PDT sessions at one-week intervals during outpatient treatment and achieved favourable results without photosensitivity and severe adverse events. Over a long follow-up period, 96.1 % of all patients showed some positive effects, including approximately 70 % with a complete response (CR), 10 % with a partial response, and 15 % with downgrades. The HPV clearance rate in patients with CR was 79.4 %. Recurrence occurred in five patients who mostly remained HPV-positive after PDT. CONCLUSIONS: Based on our study, topical 5ALA-PDT using an LED light source potentially represents a safe treatment for CIN on an outpatient basis.

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.

Photodynamic detection of a feline meningioma using 5-aminolaevulinic acid hydrochloride.

CASE SUMMARY: The present study describes the case of a feline meningioma that was detected using 5-aminolaevulinic acid hydrochloride (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence at surgery. An extra-axial mass in the temporoparietal region was observed by MRI. Following craniectomy and durotomy, photodynamic detection (PDD) was performed for detection of the tumour. Intratumour PpIX was detected using fluorescence spectrum evaluation and high-performance liquid chromatography. PDD revealed bright fluorescence of PpIX induced by 5-ALA, facilitating fluorescence-guided resection of the tumour tissue. Postoperative examination demonstrated an intratumour PpIX protein concentration of 16.8 nmol/g, and based on histopathological findings we diagnosed the mass as meningioma. RELEVANCE AND NOVEL INFORMATION: PDD using 5-ALA has been used to identify the surgical margins during resection of primary human brain tumours. Recently, we have reported post-mortem PDD using 5-ALA for a canine glioblastoma. To our knowledge, this technique has not been previously used for the detection and resection of feline brain tumours. Our findings suggest that PDD using 5-ALA is useful for intraoperative fluorescence-guided resection of malignant meningioma in cats.

5-aminolevulinic acid-mediated photodynamic diagnosis using fluorescence ureterorenoscopy for urinary upper tract urothelial carcinoma ∼Preliminary prospective single centre trial∼.

OBJECTIVE: To investigate the diagnostic accuracy of 5- aminolevulinic acid-mediated photodynamic diagnosis (ALA-PDD) for upper urinary tract tumor (UTUC) including carcinoma in situ (CIS) lesions using flexible fluorescence ureterorenoscopy. METHODS: A solution of ALA was orally administrated at 20 mg/kg body weight at 3 h prior to surgery. Fluorescence observation was carried out with IMAGE1S OPAL1 PDD system. Positive lesions that were identified as abnormal changes under white light and/or blue light were biopsied, followed by cold-cup biopsy of negative lesions that were normal-looking mucosa under white light and/or blue light. Diagnostic accuracy was analysed by comparing ureterorenoscopic observations under white light or blue light with pathological analysis results. RESULTS: A total of 31 biopsy specimens were obtained from 10 patients. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for PDD to detect UTUC was 100 %, 50 %, 52.4 % and 100 %, respectively. The sensitivity of PDD ureterorenoscopy was significantly higher than that of white light ureterorenoscopy by statistics (p < 0.05). Especially, 5 CIS lesions were detected only by ALA-PDD ureterorenoscopy (p < 0.05). Of 10 patients, one patient (10 %) experienced hypotension during the ALA-PDD. CONCLUSIONS: ALA-PDD for UTUC is a feasible and safe techniques with acceptable adverse events. Additional advantage of ALA-PDD for UTUC is the high sensitivity to detect CIS lesions with blue light mode.

Antimalarial Drugs Enhance the Cytotoxicity of 5-Aminolevulinic Acid-Based Photodynamic Therapy against the Mammary Tumor Cells of Mice In Vitro.

Artemisinin and its derivatives, including artesunate (ART) and artemether (ARM), exert anticancer effects in the micromolar range in drug and radiation-resistant cell lines. Artemisinin has been reported to sensitize cervical cancer cells to radiotherapy. In the present study, we determined whether ART and ARM could enhance the cytotoxicity of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) against the mammary tumor cells of mice. The corrected PpIX fluorescence intensities in the control, 5-ALA, 5-ALA + ART, and 5-ALA + ARM groups were 3.385 ± 3.730, 165.7 ± 33.45, 139.0 ± 52.77, and 165.4 ± 51.10 a.u., respectively. At light doses of 3 and 5 J/cm2, the viability of 5-ALA-PDT-treated cells significantly decreased with ART (p < 0.01 and p < 0.01) and ARM treatment (p < 0.01 and p < 0.01). Besides, the number of annexin V-FITC and ethidium homodimer III-positive cells was greater in the 5-ALA-PDT with ARM group than that in the other groups. N-acetylcysteine could not significantly inhibit the percentages of apoptotic cells or inviable cells induced by 5-ALA-PDT with ARM. These reactive oxygen species-independent mechanisms might enhance cytotoxicity in 5-ALA-PDT with ARM-treated tumor cells, suggesting that the use of 5-ALA-PDT with ARM could be a new strategy to enhance PDT cytotoxicity against tumor cells. However, as these results are only based on in vitro studies, further in vivo investigations are required.

5-ALA/SFC enhances HO-1 expression through the MAPK/Nrf2 antioxidant pathway and attenuates murine tubular epithelial cell apoptosis.

Cyclosporin A (CsA) is a common immunosuppressant, but its use is limited as it can cause chronic kidney injury. Oxidative stress and apoptosis play a key role in CsA-induced nephrotoxicity. This study investigated the protective effect of 5-aminolevulinic acid and iron (5-ALA/SFC) on CsA-induced injury in murine proximal tubular epithelial cells (mProx24). 5-ALA/SFC significantly inhibited apoptosis in CsA-treated mProx24 cells with increases in heme oxygenase (HO)-1, nuclear factor E2-related factor 2 (Nrf2), and p38, and Erk-1/2 phosphorylation. Moreover, 5-ALA/SFC suppressed production of reactive oxygen species in CsA-exposed cells and inhibition of HO-1 suppressed the protective effects of 5-ALA/SFC. In summary, 5-ALA/SFC may have potential for development into a treatment for the anti-nephrotoxic/apoptotic effects of CsA.

Photoirradiation after aminolevulinic acid treatment suppresses cancer cell proliferation through the HO-1/p21 pathway.

BACKGROUND: Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to control the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. Although various studies have been published regarding cell death analysis after photoirradiation by ALA-PDT, the changes in gene expressions induced by it are yet unclear. Here, we focused on studying gene expression and cell proliferation changes in cancer cells that survive photoirradiation. METHODS: HEK293 human embryonic kidney cells, MKN45 human gastric cells, and PC-3 human prostate cancer cells were selected for this research. Cell viability was measured using trypan blue and MTT assays. ALA-PDT experiments were performed using a calibrated LED irradiation module. Furthermore, mRNA and protein gene expression analysis were performed using our previously reported methods. RESULTS: mRNAs of PAI-1, HO-1, and p21 were upregulated after photoirradiation of HEK293, which was suppressed by N-acetyl-L-cysteine, a reactive oxygen species (ROS) scavenger. Primer array results in PC-3 cells and p21 and Ki-67 expression results in both PC-3 and MKN45 cells suggested that photoirradiation suppressed cell proliferation. Cell numbers post-photoirradiation revealed that the proliferation of surviving cells was suppressed in PC-3 and MKN45 cells. CONCLUSION: ALA-PDD or ALA-PDT can result in rapid ROS-induced cell death and may decrease long-term recurrence rates through several pathways including the HO-1/p21 pathway.

Effects of 5-aminolevulinic acid and sodium ferrous citrate on fibroblasts from individuals with mitochondrial diseases.

Mitochondrial respiratory chain complexes II, III, and IV and cytochrome c contain haem, which is generated by the insertion of Fe2+ into protoporphyrin IX. 5-Aminolevulinic acid (ALA) combined with sodium ferrous citrate (SFC) was reported to enhance haem production, leading to respiratory complex and haem oxygenase-1 (HO-1) upregulation. Here, we investigated the effects of different concentrations of ALA and SFC alone or in combination (ALA/SFC) on fibroblasts from 8 individuals with mitochondrial diseases and healthy controls. In normal fibroblasts, expression levels of oxidative phosphorylation (OXPHOS) complex subunits and corresponding genes were upregulated only by ALA/SFC. Additionally, the increased oxygen consumption rate (OCR) and ATP levels in normal fibroblasts were more obvious after treatment with ALA/SFC than after treatment with ALA or SFC. OXPHOS complex proteins were enhanced by ALA/SFC, whereas OCR and ATP levels were increased in 6 of the 8 patient-derived fibroblasts. Further, HO-1 protein and mRNA levels were enhanced by ALA/SFC in all fibroblasts. The relative mtDNA copy number was increased by ALA/SFC. Thus, our findings indicate that ALA/SFC is effective in elevating OXPHOS, HO-1 protein, and mtDNA copy number, resulting in an increase in OCR and ATP levels, which represents a promising therapeutic option for mitochondrial diseases.

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.

Combination of 5-aminolevulinic acid and iron prevents skin fibrosis in murine sclerodermatous graft-versus-host disease.

Scleroderma or systemic sclerosis (SSc) is a clinically heterogeneous rheumatological autoimmune disease affecting the skin, internal organs and blood vessels. There is at present no effective treatment for this condition. Our study investigated the effects of 5-aminolevulinic acid (5-ALA), which is a precursor of haem synthesis, on graft-vs-host disease (GvHD)-induced SSc murine model. Lymphocytes were intravenously injected from donor mice (B10.D2) into recipient BALB/c mice (recombination-activating gene 2 (Rag-2)-null mice) deficient in mature T and B cells to induce sclerodermatous GvHD (scl-GvHD). To investigate the effect of 5-ALA on scl-GvHD, combination of 5-ALA and sodium ferrous citrate (SFC) was orally administered to the recipient mice for 9 weeks. 5-ALA/SFC treatment significantly reduced progressive inflammation and fibrosis in the skin and ears. Furthermore, 5-ALA/SFC suppressed mRNA expression of transforming growth factor-ß, type I collagen and inflammatory cytokines. These results indicate that the 5-ALA/SFC combination treatment has a protective effect against tissue fibrosis and inflammation in a murine scl-GvHD-induced skin and ear inflammation and fibrosis. Furthermore, the efficacy of 5-ALA/SFC suggests important implications of HO-1 protective activity in autoimmune diseases, and therefore, 5-ALA/SFC may have promising clinical applications. These findings suggested that the 5-ALA/SFC treatment may be the potential strategies for SSc.

Metformin enhances the cytotoxicity of 5-aminolevulinic acid-mediated photodynamic therapy in vitro.

The biguanide metformin is a drug widely used for the treatment of type 2 diabetes. Metformin enhances the cytotoxicity of chemotherapy by promoting the adenosine monophosphate-activated protein kinase (AMPK) autophagy signaling pathway. Photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), leads to apoptosis when PpIX accumulates in the mitochondria, and also leads to autophagy through activation of AMPK. In the present study, the effect of metformin in combination with 5-ALA-PDT was evaluated in vitro in KLN205 lung cancer cells. At a fluence of 5 J/cm2, 5-ALA-PDT in combination with 5 mM metformin exhibited significantly increased cytotoxicity compared with that observed with 0 and 0.1 mM metformin (P=0.0197 and P=0.0423, respectively). The cells treated with 5-ALA-PDT and metformin exhibited condensation of nuclear chromatin and the presence of autophagosomes. These results indicate that apoptosis and autophagy occur in KLN205 cells following combined treatment with 5-ALA-PDT and metformin. The results from the present study are the first to indicate, to the best of our knowledge, that metformin potentiates the efficacy of 5-ALA-PDT.

Transposition of insertion sequence IS256Bsu1 in Bacillus subtilis 168 is strictly dependent on recA.

We developed an insertion sequence transposition detection system called the "jumping cat assay" and applied it to the Bacillus subtilis chromosome using IS256Bsu1 derived from B. subtilis natto. The high frequency of transposition enabled us to explore host factors; combining the assay and genetic analyses revealed that recA is essential for the transposition of IS256Bsu1. Detailed analyses using various domain mutants of recA demonstrated that this essentiality is not related to the function of recA in homologous recombination. Instead, the ATP binding and hydrolysis function seemed to be crucial for IS transposition. To elucidate the role of recA, we focused on the muB gene of the enterobacteriophage Mu. Based on information from the NCBI Conserved Domain Database, both MuB and RecA belong to the P-loop dNTPase superfamily. Further experiments revealed that muB complements the transposition-defective phenotype of a recA deletant, although it could not rescue UV sensitivity. These results suggest that recA shares a common function with muB that helps the transposition of IS256Bsu1 in B. subtilis.

5-Aminolevulinic acid with sodium ferrous citrate induces autophagy and protects cardiomyocytes from hypoxia-induced cellular injury through MAPK-Nrf-2-HO-1 signaling cascade.

BACKGROUND: Hypoxia causes cardiac disease via oxidative stress and mitochondrial dysfunction. 5-Aminolevulinic acid in combination with sodium ferrous citrate (ALA/SFC) has been shown to up-regulate heme oxygenase-1 (HO-1) and decrease macrophage infiltration and renal cell apoptosis in renal ischemia injury mice. However, its underlying mechanism remains largely unknown. The aim of this study was to investigate whether ALA/SFC could protect cardiomyocytes from hypoxia-induced apoptosis by autophagy via HO-1 signaling. MATERIALS & METHODS: Murine atrial cardiomyocyte HL-1 cells were pretreated with ALA/SFC and then exposed to hypoxia. RESULTS: ALA/SFC pretreatment significantly attenuated hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury, while it increased cell viability and autophagy levels. HO-1 expression by ALA/SFC was associated with up-regulation and nuclear translocation of Nrf-2, whereas Nrf-2 siRNA dramatically reduced HO-1 expression. ERK1/2, p38, and SAPK/JNK pathways were activated by ALA/SFC and their specific inhibitors significantly reduced ALA/SFC-mediated HO-1 upregulation. Silencing of either Nrf-2 or HO-1and LY294002, inhibitor of autophagy, abolished the protective ability of ALA/AFC against hypoxia-induced injury and reduced ALA/SFC-induced autophagy. CONCLUSION: Taken together, our data suggest that ALA/SFC induces autophagy via activation of MAPK/Nrf-2/HO-1 signaling pathway to protect cardiomyocytes from hypoxia-induced apoptosis.