Norio Miyoshi

Terahertz imaging diagnostics of cancer tissues with a chemometrics technique

The terahertz (THz) spectroscopic images of the paraffin embedded cancer tissues have been measured by a THz time domain spectrometer. Though the potential of the THz spectrum to the cancer diagnostics have been reported, it is sometimes difficult to distinguish normal and cancer parts due to the variety of the tissues. Thus, the systematic analysis should be introduced to clarify their difference. In this paper, the chemometrics technique has been applied to the analysis of the THz spectroscopic images of plural tumor samples. The capability of the method was discussed.

Herniation of cervical intervertebral disc: immunohistochemical examination and measurement of nitric oxide production.

Study Design. Surgically obtained cervical herniated intervertebral discs were examined histologically and immunohistochemically. The production of nitric oxide (NO) in the local tissue was examined using the electron spin resonance (ESR) method. Objectives. To investigate the local histologic and immunohistochemical changes in cervical disc herniation, including NO production, and to compare such changes with those in autopsy cases. Summary of Background Data. Very little is known about the histopathologic processes of cervical disc herniation. In addition, no information is available on the level of in vivo NO production in cervical disc herniation. Methods. Thirty-six herniated cervical discs obtained from 31 patients were immunohistochemically examined for localization of blood vessels, matrix metalloproteinase (MMP)-3, and inducible NO synthetase (iNOS). We also compared the production of NO, measured by the ESR method, in eight specimens with that of five control discs obtained from fresh cadavers. Results. The presence of herniated discs correlated with the degeneration of cartilaginous endplate and torn anulus fibrosus. Formation of new blood vessels around the herniated discs was detected, using von Willebrand factor antibody, in seven uncontained hernias and 20 contained hernias. Immunohistochemical studies showed the presence of cells positive for MMP-3 (chondrocytes), iNOS (chondrocytes and granulation tissue) in cervical disc hernias. ESR analysis showed a significantly higher NO production in herniated cervical discs than in disc samples of fresh cadavers. Conclusions. Herniated cervical intervertebral disc is characterized by the presence of an inflammatory process associated with neovascularization and increased expression of MMP-3. Production of NO was markedly high in both contained- and uncontained-type hernias.

Fluorescence-guided resection of metastatic brain tumors using a 5-aminolevulinic acid-induced protoporphyrin IX: pathological study

We performed a pathological study to identify the locus of production of protoporphyrin IX (PPIX) in human metastatic brain tumors. Patients with metastatic brain tumors (n = 11) received 1 g of 5-aminolevulinic acid (5-ALA) perorally 2 h before undergoing surgery. The target region was exposed to laser light with a peak wavelength of 405 ± 1 nm and an output of 40 mW. Tissue samples from the tumor bulk and surrounding areas were examined by histological and fluorescence methods. Of the 11 tumors, 9 manifested PPIX fluorescence in the tumor bulk and peritumoral brain tissue. Our findings indicate that PPIX fluorescence can be observed in peritumoral edematous areas that are free of neoplastic cells, because PPIX produced by neoplastic cells leaks into the surrounding edematous area.

Combination effect of photodynamic and sonodynamic therapy on experimental skin squamous cell carcinoma in C3H/HeN mice

We studied a combination of photodynamic therapy (PDT) and sonodynamic therapy (SDT) for improving tumoricidal effects in a transplantable mouse squamous cell carcinoma (SCC) model. Two sensitizers were utilized: the pheophorbide‐a derivative PH‐1126, which is a newly developed photosensitizer, and the gallium porphyrin analogue ATX‐70, a commonly used sonosensitizer. Mice were injected with either PH‐1126 or ATX‐70 i.p. at doses of 5 or 10 mg/kg.bw. At 24 (ATX‐70) or 36 hr (PH‐1126) (time of optimum drug concentration in the tumor) after injection, SCCs underwent laser light irradiation (88 J/cm2 of 575 nm for ATX‐70; 44 J/cm2 of 650 nm for PH‐1126) (PDT), ultrasound irradiation (0.51 W/cm2 at 1.0 MHz for 10 minutes) (SDT), or a combination of the two treatments. The combination of PDT and SDT using either PH‐1126 or ATX‐70 as a sensitizer resulted in significantly improved inhibition of tumor growth (92–98%) (additive effect) as compared to either single treatment (27–77%). The combination using PH‐1126 resulted in 25% of the treated mice being tumor free at 20 days after treatment. Moreover, the median survival period (from irradiation to death) of PDT + SDT‐treated mice (>120 days) was significantly greater than that in single treatment groups (77–95 days). Histological changes revealed that combination therapy could induce tumor necrosis 2–3 times as deep as in either of the single modalities. The combination of PDT and SDT could be very useful for treatment of non‐superficial or nodular tumors.

Sonodynamic toxicity of gallium-porphyrin analogue ATX-70 in human leukemia cells

Low concentrations (> or = 1 microM) of the gallium-porphyrin analogue ATX-70 significantly enhanced cellular toxicity in human leukemia HL-525 cells exposed to 50 kHz ultrasound. The mechanism of this ATX-70-dependent sonosensitization is unknown, but we have established the requirement of extracellular localization of ATX-70 molecules for sonosensitization. Short-lived toxic intermediates produced from ATX-70 by ultrasound are implicated in the mechanism, since no cytotoxicity was found when medium containing ATX-70 was sonicated and subsequently added to the cells. However, we were unable to demonstrate the existence of radical intermediates by EPR spin trapping with the nitroso spin trap, DBNBS, and ATX-70-dependent sonotoxicity could not be ameliorated by the addition of up to 70 mM POBN and DMPO spin traps during ultrasound exposure.

Terahertz pulsed imaging of frozen biological tissues

Recently, terahertz (THz) wave imaging has been shown to have potential in medical and biological applications. However, absorption by liquid water in tissues hinders the measurement of thick samples. In this study, porcine tissue was frozen to temperatures below −33 °C to prevent this absorption. Consequently, the striated muscle and adipose tissue could be clearly distinguished in the THz time-domain spectra owing to the difference in absorbance values and refractive indices. We demonstrated two-dimensional map of absorbance and THz pulse delay which clearly shows the spatial distribution of the tissues.

Effect of gallium-porphyrin analogue ATX-70 on nitroxide formation from a cyclic secondary amine by ultrasound : on the mechanism of sonodynamic activation

Sonodynamic therapy is a promising new modality for cancer treatment based on the synergistic effect on tumor cell killing by combination of a drug (typically a photosensitizer) and ultrasound. The mechanism of sonodynamic action was suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. In this work we studied the aqueous sonochemical reactions of the gallium-porphyrin derivative ATX-70, one of the most active sonodynamic agents found, using 50 kHz ultrasound. The experiments were carried out in the presence of 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TMP), which reacts with singlet oxygen or .OH radicals to give the EPR-detectable nitroxide 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TMP-NO). Recently it has been suggested that the enhancement of TMP-NO yields in the presence of aqueous solutions of ATX-70 exposed to ultrasound was evidence for the formation of singlet oxygen in the system. Our results show that the surfactant cetyltrimethylammonium bromide (CTAB) can mimic the ATX-70-induced increase in the TMP-NO signal, but it fails to reproduce the behavior of ATX-70 in D2O: while the yields of TMP-NO in the presence of ATX-70 increase in D2O, the opposite effect was found with the surfactant CTAB. However, our data show that the increased TMP-NO yields in D2O are paralleled by an increased concentration of ATX-70 dimer, a form that is inactive in the photochemical generation of singlet oxygen. Our finding that the ATX-70-dependent enhancement of the TMP-NO signal was highest at approximately 20% O2, in both N2/O2 and argon/O2 mixtures, and decreased with increasing oxygen concentration is not compatible with the singlet oxygen mechanism. Finally, our results on the temperature dependence of the ATX-70-induced formation of TMP-NO are not consistent with the photochemical excitation of ATX-70 by sonoluminescent light: the ATX-70-dependent enhancement of TMP-NO signal increased with temperature in the range 10-25 degrees C, while the intensity of sonoluminescence of aqueous solutions both in multiple-bubble fields and in single-bubble experiments is known to decrease with increasing temperature.

Enhancement of 5-Aminolevulinic acid-induced oxidative stress on two cancer cell lines by gold nanoparticles.

Abstract 5-Aminolevulinic acid (5-ALA) and its methyl ester (5-ALA-Me) at mM concentration levels induce oxidative stress via the production of reactive oxygen species (ROS). Human cancer cell lines (MCF-7 and HepG2) incubated in the dark in the simultaneous presence of 5.0 mM or more 5-ALA or 5-ALA-Me (for MCF-7) and 7 µg/mL of 15 nm citrate capped gold nanoparticles (AuNPs) were damaged more seriously compared to those in the presence of the levulinic acid alone. Damage is visible in electron micrographs which reveal similar morphology both in the presence or absence of AuNPs. Cytotoxicity was observed irrespective of the presence of serum and medium. Production of ROS in cell free samples containing 5-ALA-Me was monitored by EPR as the DMPO-OH spin adduct and also showed a catalytic effect of AuNPs. Both SOD and CAT inhibited the production of ROS and also reduced cytotoxicity in the cell samples. These observations can be explained by initial attack on the cell membrane by ROS produced in the medium outside the cell and provide insight into possible uses of 5-ALA in cancer chemotherapy.

Correlation between sonochemistry of surfactant solutions and human leukemia cell killing by ultrasound and porphyrins

The synergistic effect of ultrasound and drugs on cells is known as sonodynamic therapy. The use of sonodynamic therapy for the potential clinical treatment of certain tumors is promising, however, the mechanism of sonodynamic therapy could be due to either sonomechanical and/or sonochemical effects on the cells. The aim of the current study is to determine the importance of the sonochemical mechanism for sonodynamic therapy. Sonochemical effects arise from the formation of radical species following collapse of cavitation bubbles. The synergistic effect of ultrasound (47 kHz) and analogues of a gallium-porphyrin derivative (ATX-70) on cytolysis of Human leukemia cells (HL-525 and HL-60) suspended in a cell culture medium were studied. Organic surfactants preferentially accumulate and subsequently decompose at the gas/solution interface of cavitation bubbles, producing secondary radicals that can diffuse to the bulk solution. The gallium porphyrin analogues used in the current study possess two n-alkyl side chains (ATX-C(x), where x = number of carbon atoms, ranging from x = 2 to x = 12). By varying the n-alkyl chain length, thereby modifying the surfactant properties of the ATX-C(x) derivatives, cell killing in relation to the accumulation of ATX-C(x) derivatives at the gas/solution interface of cavitation bubbles was determined. Following sonolysis in the presence of ATX-C(x), a strong correlation for the yield of carbon-centered radicals and cell killing was observed. These results support the hypothesis that a sonochemical mechanism is responsible for the synergistic effect of ultrasound and ATX-C(x) on HL-525 and HL-60 cells.

Formation and control of two-dimensional deoxyribonucleic acid network

Recently, we have successfully fabricated large-scale deoxyribonucleic acid (DNA) networks on mica surfaces using a simple fabrication method. This report describes how we fabricated a variety of structures depending on the type of DNA, and controlled these structures using a post-treatment ethanol which we observed using atomic force microscopy. We found that the height of fiber in the DNA network depended on the type of DNA and its original length, and that the fiber height and mesh diameter could be shortened and widened, respectively, with ethanol treatment.