Table 1.
Sequences of primers used in real-time PCR.
Fig 1.
Generality of PpIX reduction and CPIII excretion after ALA administration under hypoxic conditions.
(A-D) Each cell line was incubated with 1 mM ALA for 24 h under atmospheres containing 21% O2 or 1% O2. (A) Intracellular PpIX, (B) intracellular CPIII, (C) extracellular PpIX, and (D) extracellular CPIII were measured using HPLC. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments.
Fig 2.
Oxygen dependence of PpIX reduction and CPIII excretion under hypoxic conditions.
(A-D) KatoIII gastric cancer cells were incubated with 1 mM ALA for 24 h under atmospheres containing 21%, 10%, 7.5%, 5%, 2.5%, or 1% O2. (A) Intracellular PpIX, (B) intracellular CPIII, (C) extracellular PpIX, and (D) extracellular CPIII were measured using HPLC. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments.
Fig 3.
Transcriptional regulation is not associated with the alteration of porphyrin biosynthesis during hypoxia.
(A) MKN45, KatoIII, and MKN74 cells were incubated for 24 h under atmospheres containing 21% O2 or 1% O2. mRNA expression levels of ALAD, HMBS, UROS, UROD, CPOX, PPOX, FECH, PEPT1, ABCB6, and ABCG2 in each cell line were analyzed by quantitative PCR. Data are expressed as the means ± S.D. from multi-replicated (n = 2) experiments. ALAD, aminolevulinic acid dehydrogenase; HMBS, hydroxymethylbilane synthase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen III decarboxylase; CPOX, coproporphyrinogen III oxidase; PPOX, protoporphyrinogen oxidase; FECH, ferrochelatase; PEPT1, Peptide transporter 1 (B) KatoIII cells were incubated with 100 μM CoCl2, 100 μM deferoxamine (DFO), or 1 mM dimethyloxaloglycine (DMOG) for 24 h under atmospheres containing 21% O2 or 1% O2. Immunoblots were carried out using antibodies against HIF-1α and actin. (C-F) KatoIII cells were incubated with 1 mM ALA and 100 μM CoCl2, 100 μM DFO, or 1 mM DMOG for 24 h under atmospheres containing 21% O2 or 1% O2. (C) Intracellular PpIX, (D) intracellular CPIII, (E) extracellular PpIX and (F) extracellular CPIII were measured using HPLC. (G-J) KatoIII cells were incubated with 1 mM ALA and 10 μg/mL cyclohexamide. (G) Intracellular PpIX, (H) intracellular CPIII, (I) extracellular PpIX and (J) extracellular CPIII were measured using HPLC. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments.
Fig 4.
Mitochondrial PpIX production was reduced under hypoxic conditions.
(A) KatoIII mitochondria were isolated using extraction buffer. Immunoblots were carried out using antibodies against HSP90 and COX IV-1. (B) Mitochondrial PpIX production was measured using isolated mitochondria and CPgenIII-containing medium. CPgenIII-containing medium was prepared by incubating KatoIII cells with 1 mM ALA for 24 h under an atmosphere containing 0.1% O2. CPgenIII-containing medium were added to isolated mitochondria and the mixtures were incubated for 12 h under atmospheres containing 21% O2 or 0.1% O2. PpIX was measured using HPLC. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments.
Fig 5.
Effect of respiration inhibitors on PpIX production and photodynamic therapy.
(A-D) KatoIII cells were incubated with 1 mM ALA and 0.1 μM oligomycin, 1 μM rotenone, or 1 μM antimycin for 24 h under atmospheres containing 21% O2 or 1% O2. (A) Intracellular PpIX, (B) intracellular CPIII, (C) extracellular PpIX, and (D) extracellular CPIII were measured using HPLC. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments. (E) KatoIII cells were incubated with 0.1 μM oligomycin for 24 h under atmospheres containing 21% O2 or 1% O2. mRNA expression levels of ALAD, HMBS, UROS, UROD, CPOX, PPOX, FECH, PEPT1, ABCB6, and ABCG2 were analyzed by quantitative PCR. Data are expressed as the means ± S.D. from multi-replicated (n = 2) experiments. ALAD, aminolevulinic acid dehydrogenase; HMBS, hydroxymethylbilane synthase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen III decarboxylase; CPOX, coproporphyrinogen III oxidase; PPOX, protoporphyrinogen oxidase; FECH, ferrochelatase; PEPT1, Peptide transporter 1 (F) KatoIII cells were incubated with 1 mM ALA and 0.1 μM Oligomycin for 24 h under atmospheres containing 21% O2 or 1% O2. Exposure to light was performed under an atmosphere containing 21% O2 and cell viability was measured using the MTT assay. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments.