Shun-ichiro Ogura

Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line.

Background Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. Methodology/Principal Findings Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. Conclusions/Significance The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.

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.

Preparation of a water-soluble fluorinated zinc phthalocyanine and its effect for photodynamic therapy

An amphiphilic fluorinated phthalocyanine, zinc tetracarboxyoctafluorophthalocyanine (ZnC4F8Pc) was synthesized and characterized. Its photodynamic efficiency for HeLa cells was compared with hydrophilic zinc octacarboxyphthalocyanine (ZnC8Pc) and hydrophobic zinc hexadecafluorophthalocyanine (ZnF16Pc). ZnC4F8Pc had a remarkable photodynamic effect among the phthalocyanines used. The effect is apparently caused by the fact that ZnC4F8Pc is mainly accumulated in the hydrophobic lipid membrane and is in the photoactive monomer form in HeLa cells.

Development of phthalocyanines for photodynamic therapy

Photodynamic therapy is a method for treating several diseases, most notably cancer. Recent synthetic activity has created a number of phthalocyanines for potential use as photodynamic therapy photosensitizers. In this mini-review article, the background and the concepts in the development of new phthalocyanines are introduced.

Sugar-dependent photocytotoxic property of tetra- and octa-glycoconjugated tetraphenylporphyrins

Abstract New tetraphenylporphyrin derivatives having eight glucose molecules were synthesized. Phototoxicity of these compounds against the HeLa cell line were compared to tetraglycosylated tetraphenylporphyrins. The highest activity was observed for a derivative having four OH-protected glucose moieties. Singlet oxygen producing ability was also examined to explain the difference in photocytotoxicities.

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.

Cellular Uptake and Photocytotoxicity of Glycoconjugated Porphyrins in Hela Cells.

Thirty‐two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)2.2H2O as a template. The Zn2+ ion template strategy improved the yield about three‐fold in the case of meta ‐substituted tetraphenylporphyrins. In addition, free‐base porphyrins were obtained almost quantitatively by demetalation with 4 M HCI. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15, 20‐tetrakis[4‐(β‐D‐arabinopyranosyloxy)phenyl]porphyrin p‐5d) in particular showed 18.5‐fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20‐tetrakis[4‐(β‐D‐glucopyranosyloxy) phenyl]porphyrin (p‐5a), (p‐5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm2. These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS p‐5a p‐5d. These results suggest p‐5d is a good candidate for a PDT drug.

Photodynamic Efficiency of Protoporphyrin IX: Comparison of Endogenous Protoporphyrin IX Induced by 5-Aminolevulinic Acid and Exogenous Porphyrin IX

Abstract— Photodynamic efficiency of protoporphyrin IX (PP) accumulated in HeLa cells by the incubation of PP with HeLa cells was compared with that of accumulated PP formed from 5‐aminolevulinic acid (ALA) as a precursor. The ALA‐induced PP was photodynamically more efficient than exogenous PP. The difference is caused by monomelic PP concentration and PP localization site in HeLa cells. Exogenous PP was accumulated mainly in plasma membrane, and the membrane was strongly damaged by irradiation. The ALA‐induced PP was selectively accumulated in mitochondria and inactivated the mitochondrial function by irradiation.

Enhanced expression of coproporphyrinogen oxidase in malignant brain tumors: CPOX expression and 5-ALA–induced fluorescence

In photodynamic diagnosis, 5-aminolevulinic acid (5-ALA) is widely used for the fluorescence-guided resection of malignant brain tumors, where 5-ALA is converted to protoporphyrin IX, which exhibits strong fluorescence. Little is known, however, about the detailed molecular mechanisms underlying 5-ALA-induced fluorescence. To resolve this issue, we analyzed transcriptome profiles for the genes encoding enzymes, transporters, and a transcription factor involved in the porphyrin-biosynthesis pathway. By quantitative real-time (qRT)-PCR, we measured the mRNA levels of those genes in a total of 20 tumor samples that had been surgically resected from brain tumor patients at the Department of Neurosurgery of Osaka Medical College from 2008 to 2009. We selected 10 tumor samples with no 5-ALA-induced fluorescence, among which 2 were glioblastomas and 8 were metastatic brain tumors. Another 10 tumor samples were selected with strong fluorescence, among which 7 were glioblastomas and 3 were metastatic brain tumors. The qRT-PCR analysis study of these latter 10 samples revealed predominantly high levels of the mRNA of the coproporphyrinogen oxidase (CPOX) gene. The high mRNA level of CPOX expression was significantly well correlated with the phenotype of strong 5-ALA-induced fluorescence (P = .0003). These findings were further confirmed by immunohistochemical studies with a CPOX-specific antibody. It is concluded that induction of CPOX gene expression is one of the key molecular mechanisms underlying the 5-ALA-induced fluorescence of malignant brain tumors. The induction mechanism for the CPOX gene in brain tumors remains to be elucidated.