Kiwamu Takahashi

Novel development of 5-aminolevurinic acid (ALA) in cancer diagnoses and therapy.

Early detection and intervention are needed for optimal outcomes in cancer therapy. Improvements in diagnostic technology, including endoscopy, photodynamic diagnosis (PDD), and photodynamic therapy (PDT), have allowed substantial progress in the treatment of cancer. 5-Aminolevulinic acid (ALA) is a natural, delta amino acid biosynthesized by animal and plant mitochondria. ALA is a precursor of porphyrin, heme, and bile pigments, and it is metabolized into protoporphyrin IX (PpIX) in the course of heme synthesis. PpIX preferentially accumulates in tumor cells resulting in a red fluorescence following irradiation with violet light and the formation of singlet oxygen. This reaction, utilized to diagnose and treat cancer, is termed ALA-induced PDD and PDT. In this review, the biological significance of heme metabolites, the mechanism of PpIX accumulation in tumor cells, and the therapeutic potential of ALA-induced PDT alone and combined with hyperthermia and immunotherapy are discussed.

Pivotal roles of peptide transporter PEPT1 and ATP-binding cassette (ABC) transporter ABCG2 in 5-aminolevulinic acid (ALA)-based photocytotoxicity of gastric cancer cells in vitro

BACKGROUND Recently, 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is being widely used in cancer therapy owing to the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In the present study, by focusing on genes involved in the porphyrin biosynthesis pathway, we aimed to explore biomarkers that are predictive for the efficacy of ALA-PDT. METHODS We used five lines of human gastric cancer cells to measure the ALA-based photocytotoxicity. ALA-induced production of PpIX in cancer cells was quantified by fluorescence spectrophotometry. To examine the potential involvement of PEPT1 and ABCG2 in the ALA-PDT sensitivity, stable cell lines overexpressing PEPT1 were established and ABCG2-specific siRNA used. RESULTS We observed that three cell lines were photosensitive, whereas the other two cell lines were resistant to ALA-based photocytotoxicity. The ALA-based photocytotoxicity was found to be well correlated with intracellular PpIX levels, which suggests that certain enzymes and/or transporters involved in ALA-induced PpIX production are critical determinants. We found that high expression of the peptide transporter PEPT1 (ALA influx transporter) and low expression of the ATP-binding cassette transporter ABCG2 (porphyrin efflux transporter) determined ALA-induced PpIX production and cellular photosensitivity in vitro. CONCLUSION PEPT1 and ABCG2 are key players in regulating intracellular PpIX levels and determining the efficacy of ALA-based photocytotoxicity against gastric cancer cells in vitro. Evaluation of the expression levels of PEPT1 and ABCG2 genes could be useful to predict the efficacy of ALA-PDT. Primers specific to those target genes are practical and useful biomarkers for predicting the photo-sensitivity to ALA-PDT.

The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver.

Background5-Aminolevulinic acid (ALA) is a precursor of heme that is fundamentally important in aerobic energy metabolism. Among the enzymes involved in aerobic energy metabolism, cytochrome c oxidase (COX) is crucial. In this study, the effect of ALA on cytochrome c oxidase activity was measured.Findingsc57BL/6N species of mice were administered ALA orally for 15 weeks. After ALA administration, mice were sacrificed and livers were obtained. COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from PBS-administered mouse livers (P < 0.05). Furthermore, ATP levels in ALA-administered mouse livers were much higher than those in PBS-administered mouse livers. These data suggest that oral administration of ALA promotes aerobic energy metabolism, especially COX activity.ConclusionsThis is the first report of a drug that functions in aerobic energy metabolism directly. Since COX activity is decreased in various diseases and aging, the pharmacological effects of ALA will be expanding.

Fluorescent detection of peritoneal metastasis in human colorectal cancer using 5-aminolevulinic acid

A precise diagnosis of peritoneal dissemination is necessary to determine the appropriate treatment strategy for colorectal cancer. However, small peritoneal dissemination is difficult to diagnose. 5-aminolevulinic acid (5-ALA) is an intermediate substrate of heme metabolism. The administration of 5-ALA to cancer patients results in tumor-specific accumulation of protoporphyrin IX (PpIX), which emits red fluorescence with blue light irradiation. We evaluated the usefulness of photodynamic diagnosis (PDD) using 5-ALA to detect the peritoneal dissemination of colorectal cancer. EGFP-tagged HT-29 cells were injected into the peritoneal cavity of BALB/c nude mice. After 2 weeks, the mice were given 5-ALA hydrochloride, and metastatic nodules in the omentum were observed with white light and fluorescence images. Twelve colorectal cancer patients suspected to have serosal invasion according to preoperative computed tomography (CT) were enrolled in this study. 5-ALA (15-20 mg per kg body weight) was administered orally to the patients 3 h before surgery. The abdominal cavity was observed under white light and fluorescence. Fluorescence images were analyzed with image analysis software (ImageJ 1.45s, National Institutes of Health, Bethesda, MD, USA). The mice developed peritoneal disseminations. The observed 5-ALA-induced red fluorescence was consistent with the EGFP fluorescent-positive nodules. Peritoneal dissemination was observed with conventional white light imaging in 8 patients. All nodules suspected as being peritoneal dissemination lesions by white light observation were similarly detected by ALA-induced fluorescence. In 1 patient, a small, flat lesion that was missed under white light observation was detected by ALA-induced fluorescence; the lesion was pathologically diagnosed as peritoneal metastasis. In the quantitative fluorescence image analysis, the red/(red + green + blue) ratio was higher in the metastatic nodules compared to the non-metastatic sites of the abdominal wall, fat and liver. We demonstrated better diagnostic accuracy using 5-ALA-PDD compared to conventional laparoscopy in patients with colorectal cancer. 5-ALA-PDD is a promising candidate method for diagnosing peritoneal dissemination of colorectal cancer.

5-Aminolevulinic acid combined with ferrous iron enhances the expression of heme oxygenase-1

5-Aminolevulinic acid (5-ALA) is the naturally occurring metabolic precursor of heme. Heme negatively regulates the Maf recognition element (MARE) binding- and repressing-activity of the Bach1 transcription factor through its direct binding to Bach1. Heme oxygenase (HO)-1 is an inducible enzyme that catalyzes the rate-limiting step in the oxidative degradation of heme to free iron, biliverdin and carbon monoxide. These metabolites of heme protect against apoptosis, inflammation and oxidative stress. Monocytes and macrophages play a critical role in the initiation, maintenance and resolution of inflammation. Therefore, the regulation of inflammation in macrophages is an important target under various pathophysiological conditions. In order to address the question of what is responsible for the anti-inflammatory effects of 5-ALA, the induction of HO-1 expression by 5-ALA and sodium ferrous citrate (SFC) was examined in macrophage cell line (RAW264 cells). HO-1 expression induced by 5-ALA combined with SFC (5-ALA/SFC) was partially inhibited by MEK/ERK and p38 MAPK inhibitor. The NF-E2-related factor 2 (Nrf2) was activated and translocated from the cytosol to the nucleus in response to 5-ALA/SFC. Nrf2-specific siRNA reduced the HO-1 expression. In addition, 5-ALA/SFC increased the intracellular levels of heme in cells. The increased heme indicated that the inactivation of Bach1 by heme supports the upregulation of HO-1 expression. Taken together, our data suggest that the exposure of 5-ALA/SFC to RAW264 cells enhances the HO-1 expression via MAPK activation along with the negative regulation of Bach1.

5-Aminolevulinic acid combined with ferrous iron induces carbon monoxide generation in mouse kidneys and protects from renal ischemia-reperfusion injury

Renal ischemia reperfusion injury (IRI) is a major factor responsible for acute renal failure. An intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA) is fundamental in aerobic energy metabolism. Heme oxygenase (HO)-1 cleaves heme to form biliverdin, carbon monoxide (CO), and iron (Fe(2+)), which is used with 5-ALA. In the present study, we investigated the role of 5-ALA in the attenuation of acute renal IRI using a mouse model. Male Balb/c mice received 30 mg/kg 5-ALA with Fe(2+) 48, 24, and 2 h before IRI and were subsequently subjected to bilateral renal pedicle occlusion for 45 min. The endogenous CO concentration of the kidneys from the mice administered 5-ALA/Fe(2+) increased significantly, and the peak concentrations of serum creatinine and blood urea nitrogen decreased. 5-ALA/Fe(2+) treatments significantly decreased the tubular damage and number of apoptotic cells. IRI-induced renal thiobarbituric acid-reactive substance levels were also significantly decreased in the 5-ALA/Fe(2+) group. Furthermore, mRNA expression of HO-1, TNF-α, and interferon-γ was significantly increased after IRI. Levels of HO-1 were increased and levels of TNF-α and interferon-γ were decreased in the 5-ALA/Fe(2+)-pretreated renal parenchyma after IRI. F4/80 staining showed reduced macrophage infiltration, and TUNEL staining revealed that there were fewer interstitial apoptotic cells. These findings suggest that 5-ALA/Fe(2+) can protect the kidneys against IRI by reducing macrophage infiltration and decreasing renal cell apoptosis via the generation of CO.

Access to a novel near-infrared photodynamic therapy through the combined use of 5-aminolevulinic acid and lanthanide nanoparticles

BACKGROUND There have been considerable efforts to develop photodynamic therapy (PDT) for cancer, in which photoirradiation of a sensitizer delivered near cancer cells results in the conversion of oxygen into active species, causing cell destruction. Aiming at the best cancer selectivity, one PDT method employed protoporphyrin IX (PPIX), which selectively accumulated in cancer cells after oral administration of 5-aminolevulinic acid (ALA). The drawback, however, is that blue incident lights are required to excite PPIX, resulting in low tissue penetrability, and therefore limiting its application to surface cancers. METHODS To overcome the low penetrability of the incident light, we employed a light energy upconverter, lanthanide nanoparticle (LNP), which, upon irradiation with highly penetrative near-infrared (NIR) radiation, emits visible light within the Q-band region of PPIX absorbance allowing its sensitization. To discover the optimum conditions for the LNP-assisted PDT, the cytotoxicity and PPIX-sensitizability of LNPs were first studied. Then, the LNP-assisted PDT was validated using the MKN45 cell line: cells were pretreated with ALA and LNP, irradiated with a 975-nm diode laser, and subjected to MTT assay to measure cell viability. RESULTS The singlet oxygen generation on NIR-irradiation of the PPIX-LNP mixture was proved, indicating that the emission from LNP could excite the PPIX sensitizer. An intermittent NIR-irradiation for 32 min of MKN45, pretreated with LNP (1mg/mL) and ALA (2mM), caused 87% cell destruction. CONCLUSIONS The potential applicability of the NIR-irradiation PDT with ALA- and LNP-pretreated cancer cells was demonstrated.

Porphyrins in urine after administration of 5-aminolevulinic acid as a potential tumor marker.

BACKGROUND Tumor markers are commonly used for cancer screening and as indicators of therapeutic effects. Certain types of tumor have been known to produce a variety of porphyrins after 5-aminolevulinic acid (ALA) administration. In this study, porphyrins in tumor-bearing mouse urine were analyzed after oral administration of ALA in order to identify new tumor markers excreted in the urine. METHODS Porphyrin concentrations in the urine of tumor-bearing mice were measured after administration of 1.0mg of ALA (approximately 50mgkg(-1)). RESULTS Porphyrin concentrations in the urine of tumor-bearing mice increased after administration of ALA. HPLC analysis of the urine revealed the existence of uroporphyrin (UP) and coproporphyrin (CP) in the urine of ALA-treated tumor-bearing mice. Furthermore, at 3h after ALA administration, UP concentrations in the urine of tumor-bearing mice significantly increased compared to those in the urine of normal mice. CONCLUSION These results suggest that UP as a precursor of heme detected in the urine of tumor-bearing mice after ALA administration is a potential marker of tumor development.

Functional myo-inositol catabolic genes of Bacillus subtilis Natto are involved in depletion of pinitol in Natto (fermented soybean).

Soybeans are rich in pinitol (PI; 3-O-methyl-D-chiro-inositol), which improves health by treating conditions associated with insulin resistance, such as diabetes mellitus and obesity. Natto is a food made from soybeans fermented by strains of Bacillus subtilis natto. In the chromosome of natto strain OK2, there is a putative promoter region almost identical to the iol promoter for myo-inositol (MI) catabolic genes of B. subtilis 168. In the presence of MI, the putative iol promoter functioned to induce inositol dehydrogenase, the enzyme for the first-step reaction in the MI catabolic pathway. PI also induced inositol dehydrogenase and the promoter was indispensable for the utilization of PI as well as MI, suggesting that PI might be an alternative carbon source metabolized in a way involving the MI catabolic genes. Natto fermentation studies have revealed that the parental natto strain consumed PI while a mutant defective in the iol promoter did not do so at all. These results suggest that inactivating the MI catabolic genes might prevent PI consumption, retaining it in natto for enrichment of possible health-promoting properties.

5-Aminolevulinic acid with ferrous iron induces permanent cardiac allograft acceptance in mice via induction of regulatory cells

BACKGROUND 5-Aminolevulinic acid (5-ALA), a precursor of heme biosynthesis, plays a fundamentally important role in aerobic energy metabolism. Heme oxygenase (HO)-1 cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe(2+)). The anti-inflammatory properties of biliverdin and CO help to alleviate ischemia/reperfusion injury as well as acute and/or chronic allograft rejection. We investigated whether 5-ALA and Fe(2+) exerts salutary effects in the setting of organ transplantation. METHODS An in vitro mixed-lymphocyte reaction (MLR) assay and cardiac allotransplantation model (CBA to C57BL/10) were used to evaluate the effects of 5-ALA and Fe(2+) on transplantation tolerance. RESULTS Treatment with 5-ALA and sodium ferrous citrate (SFC) resulted in permanent acceptance in the murine cardiac allografts in a dose-, SFC- and HO-1-dependent manner. The number of graft-infiltrating CD8 T cells was lower and the survival response of recipient spleen T cells to donor-type alloantigens was less compared with control recipients; however, numbers of both regulatory T cells and dendritic cells were significantly increased in 5-ALA/SFC-treated recipients. CONCLUSIONS Our findings show that 5-ALA/SFC inhibits T-cell proliferation in response to alloantigens and an increased number of regulatory cells, resulting in permanent cardiac allograft acceptance in mice. These findings highlight the major roles of CO and/or HO-1 in inducing tolerance and suggest that 5-ALA/SFC may be a clinically effective treatment for allograft rejection.