Chin-Tin Chen

Improved photodynamic inactivation of gram-positive bacteria using hematoporphyrin encapsulated in liposomes and micelles

Antimicrobial photodynamic inactivation (PDI) is a promising treatment modality for local infections. To increase the efficacy of photosensitizer, hematoporphyrin (Hp) was used as a model drug and encapsulated in liposomes and micelles. The bactericidal efficacy of the carrier‐entrapped Hp was assessed against gram‐positive bacteria.

Diagnosis of oral cancer by light-induced autofluorescence spectroscopy using double excitation wavelengths

A cancer diagnostic algorithm, light-induced autofluorescence spectroscopy using double excitations wavelengths, was employed for distinguishing between cancerous and normal oral mucosa. For emission spectra at the shorter excitation wavelengths (280, 290, and 300 nm), the ratio between the area under 325-335 nm and the area under 465-475 nm was calculated. In the same way, for emission spectra at the longer excitation wavelengths (320, 330, and 340 nm), the ratio between the area under 375-385 nm and the area under 465-475 nm was calculated. Receiver operating characteristic curves were used to evaluate the performance of algorithms using single and the double (by combining shorter and longer) excitation wavelengths. The results showed that better performance, up to sensitivity 81.25%, specificity 93.75%, and positive predictive value 92.86%, could be achieved by using the double excitation wavelengths. The present study can be useful as a basis for further investigation on in vivo autofluorescence measurement and analysis using double excitation wavelength.

ALA-PDT results in phenotypic changes and decreased cellular invasion in surviving cancer cells.

The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. This study attempted to investigate the influence of 5‐aminolevulinic acid (ALA)‐based PDT on the invasion ability as well as molecular changes in surviving cancer cells and their progeny.

A PROBABILITY-BASED MULTIVARIATE STATISTICAL ALGORITHM FOR AUTOFLUORESCENCE SPECTROSCOPIC IDENTIFICATION OF ORAL CARCINOGENESIS

Abstract— A probability‐based multivariate statistical algorithm combining partial least‐squares (PLS) and logistic regression was developed to identify the development stages of oral cancer through analysis of autofluorescence spectra of oral tissues. Tissues were taken from a 7, 12‐dimethyl‐benz[a]anthracene‐induced hamster buecal pouch carcinogenesis model. Analyses were conducted at various excitation wavelengths, ranging from 280 nm to 400 nm in 20 nm increments, to assess classification performance at different excitations. For each excitation the PLS analysis and logistic regression were combined, on the basis of cross validation, to calculate the posterior probabilities of samples belonging to four stages of cancer development: normal tissues, hyperplasia, dysplasia and early cancers and frankly invasive cancers. Results showed that the 320 nm excitation wavelength optimally classified the cancer development stages: the accuracy rates for identifying samples at that excitation were 91.7%, 83.3%, 66.7% and 83.3% for the four respective stages. The average accuracy rate was 81.3%. These results suggest that the algorithm described in this study might be useful for the detection of human oral cancers.

G-Quadruplex Stabilizer 3,6-Bis(1-Methyl-4-Vinylpyridinium)Carbazole Diiodide Induces Accelerated Senescence and Inhibits Tumorigenic Properties in Cancer Cells

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13°C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 μmol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated β-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways. (Mol Cancer Res 2008;6(6):955–64)

Toluidine Blue O Photodynamic Inactivation on Multidrug-Resistant Pseudomonas Aeruginosa

Multidrug‐resistant (MDR) Pseudomonas aeruginosa infection is becoming a critical problem worldwide. Currently, only limited therapeutic options are available for the treatment of infections caused by MDR P. aeruginosa, therefore, the development of new alternative treatments is needed. Toluidine blue O (TBO) is an effective antibacterial photosensitizing agent against various bacteria. However, reports on antibacterial photosensitization of MDR bacteria are limited. This study aims to determine the in vitro photobactericidal activity of TBO against MDR P. aeruginosa.

5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase

Photodynamic therapy (PDT) has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) signaling cascades are activated following 5-aminolevulinic acid (ALA)-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.

Comparative study on the ALA photodynamic effects of human glioma and meningioma cells

The purpose of this study was to compare the differential susceptibility to photodynamic therapy (PDT) mediated damage in human U‐105MG glioma cells and CH‐157MN meningioma cells in vitro using 5‐aminolevulinic acid (ALA) as photosensitizer, and to determine if growth factors would enhance PDT‐mediated damage of these cells.

Chitosan Augments Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

ABSTRACT Antimicrobial photodynamic inactivation (PDI) was shown to be a promising treatment modality for microbial infections. This study explores the effect of chitosan, a polycationic biopolymer, in increasing the PDI efficacy against Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and methicillin-resistant S. aureus (MRSA), as well as the Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. Chitosan at <0.1% was included in the antibacterial process either by coincubation with hematoporphyrin (Hp) and subjection to light exposure to induce the PDI effect or by addition after PDI and further incubation for 30 min. Under conditions in which Hp-PDI killed the microbe on a 2- to 4-log scale, treatment with chitosan at concentrations of as low as 0.025% for a further 30 min completely eradicated the bacteria (which were originally at ∼108 CFU/ml). Similar results were also found with toluidine blue O (TBO)-mediated PDI in planktonic and biofilm cells. However, without PDI treatment, chitosan alone did not exert significant antimicrobial activity with 30 min of incubation, suggesting that the potentiated effect of chitosan worked after the bacterial damage induced by PDI. Further studies indicated that the potentiated PDI effect of chitosan was related to the level of PDI damage and the deacetylation level of the chitosan. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.

Skin denervation, neuropathology, and neuropathic pain in a laser-induced focal neuropathy

Small-diameter sensory nerves innervating the skin are responsive to noxious stimuli, and an injury to these nerves is presumably related to neuropathic pain. Injury-induced neuropathic pain in animals can be produced by laser irradiation, which usually requires concomitant use of photosensitive dyes, known as the photochemical approach. It is not clear whether laser irradiation alone can induce neuropathic pain. In addition, two issues are important to apply these approaches: the relationship between the extent of laser irradiation and the occurrence of neuropathic pain, and the susceptibility of small-diameter sensory nerves in the skin to laser-induced neuropathic pain. To address these issues, we designed a new model of focal neuropathy by applying a diode laser of 532 nm (100 mW) to the sciatic nerve and evaluated small-diameter nerves by quantifying skin innervation and large-diameter nerves by measuring amplitudes of the compound muscle action potential (CMAP). Immediately after laser irradiation, epineurial vessels were occluded due to the formation of thrombi, and the blood flow through these vessels was markedly reduced. On postoperative day (POD) 2, animals developed characteristic manifestations of neuropathic pain, including spontaneous pain behaviors, thermal hyperalgesia, and mechanical allodynia. These phenomena peaked during PODs 7-21, and lasted for 3-6 weeks. The neuropathology at the irradiated site of the sciatic nerve included a focal area of axonal degeneration surrounded by demyelination and endoneurial edema. The extent of damage to large-diameter motor and sensory nerves after laser irradiation was evaluated by nerve conduction studies. On the irradiated sides, amplitudes of the compound muscle action potentials and sensory nerve action potentials (SNAPs) were reduced to 65.0% (P < 0.0001) and 42.5% (P < 0.01) of those on the control sides, respectively. Motor innervation of the neuromuscular junctions (NMJs) on plantar muscles was examined by combined cholinesterase histochemistry and immunohistochemistry. The ratio of innervated NMJs on the operated sides decreased to 76.3% of that on the control side. Skin innervation in the territory of the irradiated sciatic nerves was evaluated by immunohistochemistry with neuronal markers. Among these markers, epidermal nerve densities for protein gene product (PGP) 9.5, calcitonin gene-related peptide (CGRP), and substance P (SP) were significantly lower on the irradiated sides than the control sides with a different degree of loss for each marker (42.1-53.1%, P < 0.05). Results suggest that laser-induced focal neuropathy provides a new system for studying neuropathic pain. With this approach, the extent of nerve injury can be quantified. Both small-diameter epidermal nerves and large-diameter sensory and motor nerves are susceptible to laser-induced injury of different degrees.