Enhancement of cytotoxic effect on human head and neck cancer cells by combination of photodynamic therapy and sulforaphane.

Photodynamic therapy (PDT) is a method to treat cancers using photosensitizer and light. PDT has been tried for several tumors. However, the clinical applications are limited by the toxicity of photosensitizer and narrow effect. Sulforaphane (SFN) is a material of isothiocyanate group and known to have anticancer effect. We evaluated the cytotoxic effect of PDT combined with SFN on human head and neck cancer cells. We measured the cell viability, extent of apoptosis and necrosis, reactive oxygen species (ROS) generation and caspase activation. Cell viability was decreased significantly by combination treatment. Cellular apoptosis and necrosis were increased in combination treatment compared to SFN or PDT. ROS generation was also higher in combination treatment than single treatment. In combination treatment group, apoptosis and necrosis were decreased by administration of sodium azide (SA) which is scavenger of ROS. Increased caspase activation in combination treatment was also inhibited by SA. Combination of PDT and SFN led to enhanced cytotoxic effect on head and neck cancer cells. Combination treatment promoted the ROS generation, which induced cell death through activation of caspase pathway.

Combination treatment of Cetuximab and photodynamic therapy in SNU-1041 squamous cancer cell line.

Cetuximab (Erbitux) has been highlighted for its anti-proliferative effects in solid tumors and it is currently used as an adjuvant modality with other anti-cancer treatments. Photodynamic therapy (PDT) is used widely in many specialties of medicine. This study evaluated the efficacy of a combination treatment of two modalities (Cetuximab, PDT) both in vivo and in vitro. The SNU-1041 cell line was used for both the in vitro and in vivo studies. The in vivo and in vitro experiments were each classified into four groups, control group, Cetuximab applied group, PDT applied group and combined modality group. A migration study was performed to determine the anti-migration effect of Cetuximab, and a MTT assay was performed to compare the anti-proliferative effect of the modalities in vitro. For the in vivo study, the cells were implanted into 5-week-old nude mice. The measured volume of the tumor for each group was compared as a function of time. In the migration study, the control group showed a longer migration length than the Cetuximab applied group. In the MTT assay, the combination modality group showed less survival than the uni-modality groups. The measured tumor size after treatment showed that the combination treatment was more effective than the single modalities. PDT and Cetuximab are treatment modalities that target different molecular pathways. A combination of these two treatment modalities was found to more effective than an individual treatment. However, further studies will be needed to determine the optimal dose of the photosensitizer and Cetuximab.

Photodynamic therapy with 9-hydroxypheophorbide alpha on AMC-HN-3 human head and neck cancer cells: induction of apoptosis via photoactivation of mitochondria and endoplasmic reticulum.

Skin phototoxicity is one of the main side effects of photodynamic therapy (PDT). To overcome this problem, some new photosensitizers have been developed with longer absorbance wavelengths and shorter half-life in the body. In this study, we investigated the mechanism of PDT mediated by a new chlorophyll derivative photosensitizer, 9-hydroxypheophorbide alpha (9-HPbD), on AMC-HN-3 cancer cells. Phototoxicity and apoptosis on AMC-HN-3 cells induced by 9-HPbD was exhibited in a time- and dose-dependent manner. Mitochondria and endoplasmic reticulum (ER) were observed as preferential sites of 9-HPbD accumulation. Photoactivation of 9-HPbD-loaded AMC-HN-3 cells led to a rapid generation of reactive oxygen species (ROS) at 30 min, followed by a loss of mitochondrial membrane potential (MMP) at 2 h, translocation of apoptosis-inducing factor (AIF) at 2 h, and the release of cytochrome c at 3 h following PDT. Caspase-12, an important caspase involved in ER-induced apoptosis, and C/EBP homologous protein (CHOP), an ER stress inducible transcription factor, were also upregulated after PDT (3-12 h and 6-12 h, respectively). Subsequently, activation of caspase-9 at 6 h, caspase-3 and PARP at 12 h also occurred in PDT-treated AMC-HN-3 cells. The above observations demonstrate that both mitochondria and ER serve not only as the sites of sensitizer binding, but also the subcellular targets of 9-HPbD-PDT, effective activation of which is responsible for 9-HPbD PDT-induced apoptosis in AMC-HN-3 cells.

Antioxidant and anti-apoptotic effect of melatonin on the vestibular hair cells of rat utricles.

OBJECTIVES: Aminoglycosides are commonly used antibiotic agents, and they are known to generate free oxygen radicals within the inner ear and to cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Melatonin, a pineal secretory product, has the properties of being a powerful direct and indirect antioxidant. The aim of the present study was to prove the antioxidant effect of melatonin against gentamicin-induced ototoxicty. METHODS: The utricular maculae of Sprague-Dawley rats were prepared from postnatal day 2-4, and these maculae were were divided into 6 groups as follows: 1) control, 2) melatonin only, 3) gentamicin only, and 4), 5), and 6) gentamicin plus melatonin (10, 50, and 100 microM, respectively). To count the number of hair cells, 5 utricles from each group were stained with phalloidin-FITC on the 1st, 4th, and 7th days after drug administration. Reactive oxygen species (ROS) was assessed by using the fluorescent probe hydrofluorescent diacetate acetyl ester. The caspase-3 activity was also examined with using the fluorescent caspase-3 substrate and performing Western blotting. RESULTS: The result of this study showed that gentamicin induced the loss of utricular hair cells, and this loss of hair cells was significantly attenuated by co-administration of melatonin. Melatonin reduced ROS production and caspase-3 activation in the gentamicin treated utricular hair cells. CONCLUSION: Our findings conclusively reveal that melatonin has protective effects against gentamicin-induced hair cell loss in the utricles of rat by inhibiting both ROS production and caspase-3 activity.

Enhanced apoptotic effect of combined modality of 9-hydroxypheophorbide alpha-mediated photodynamic therapy and carboplatin on AMC-HN-3 human head and neck cancer cells.

Photodynamic therapy (PDT) has been developed as an effective treatment for malignant disease. Carboplatin (CBDCA), a less nephrotoxic analog of cisdiamminedichloroplatinum (cisplatin), has been widely used for the treatment of multiple malignancies. In this study, we investigated the cytotoxic and apoptotic effect of combined modality of 9-hydroxypheophorbide alpha (9-HPbD)-mediated PDT and CBDCA on AMC-HN-3 human head and neck cancer cell line in vitro. The attached AMC-HN-3 cells were incubated with CBDCA (0.04 mg/ml) for 24 h at 37 degrees C and followed by photosensitization with 9-HPbD for 6 h and laser irradiation with 670 nm diode laser at an intensity of 2.0 J/cm(2) for activating 9-HPbD for 15 min. Then MTT reduction assay and Hoechst 33342 and propidium iodide (PI) double staining were used respectively to measure the cytotoxicity and nuclear morphology at 24 h after PDT. Expression of caspase-3, -9 and poly(ADP-ribose) polymerase (PARP) was detected at 0, 3, 6 and 12 h after irradiation through Western blotting techniques. Compared with PDT and CBDCA alone groups, there was more cytotoxicity and enhanced apoptotic cell death in combination groups. The peaked expression of cleaved form of caspase-3, -9 and PARP occurred approximately 3 h after PDT. There was stronger expression of cleaved caspase-3, -9 and PARP in combination groups than that in PDT or CBDCA alone groups. This study demonstrates that the combined modality resulted in enhanced apoptotic cell death as well as cytotoxic effect on AMC-HN-3 cells in vitro, which suggests the feasibility of combined modality and the possibility of reducing the effective dosage of 9-HPbD and CBDCA and lowering the side effects on normal cells.