In vivo study of photodynamic therapy using a new photosensitizer, 9-hydroxypheophorbide-a and 670 nm diode laser on HT-3 cervical cancer cell / 대한산부인과학회지

OBJECTIVE: During the last several years, such as Photofrin(R), Photogem(R), HpD, and 5-aminolevulenic acid (ALA) has been used as photosensitizers for a wide range of malignant tumors as well as non-malignant disease. A new photosensitizer, 9-Hydroxypheophorbide-a (9-HpbD-a) was derived from Spirulina platensis. The aim of this study was to evaluate the anticancer effect and mechanism of photodynamic therapy in vivo using 9-HpbD-a and 670 nm diode laser on a HT-3 cervical cancer cell line. METHODS: PDT effects were observed in xenograft nude mouse model. HT-3 cell line was xenografted into the subcutaneous space of the mouse back. When the tumors grew up to 400~500 mm3, the animals were randomly separated into 4 groups: Group I (n=5), control group; group II (n=10), 9-HpbD-a alone; group III (n=10), laser irradiation alone; and group IV (n=20), PDT in nude mouse group. RESULTS: PDT treated group (9-HpbD-a+laser irradiation) showed complete response (15/20, 75%), recurrence (3/20, 15%) or no response (2/20, 10%). PDT group showed significant remission rate, comparing to laser irradiation alone group, 9-HpbD-a alone group and untreated control group (P<0.05). When PDT using 9-HpbD-a, tumor size was decrease and microscopically finding, antitumor effect was showed inflammation and necrosis of tumor cell. Overexpression of p53 and Ki-67 gene had been detected at the HT-3 cervical cancer cell and 10 days after PDT. And no experssion in normal tissue and tumor after PDT. So, immunohistochemical study of p53 and Ki-67 gene correlated with PDT for anticancer effect. CONCLUSION: These results suggest that 9-HpbD-a is one of the promising photosensitizers for the future and would be needed to use 9-HpbD-a as a photosensitizer in clinical setting. We would further suggest that photodynamic therapy is a more effective method than the conventional treatment for women who want to maintain their anatomical and functional uterine cervix.

The anticancer effect and mechanism of photodynamic therapy using a new photosensitizer and diode laser on cervical cancer cell / 대한산부인과학회지

OBJECTIVE: During the last several years, such as Photofrin(R), Photogem(R), HpD, and 5-aminolevulenic acid (ALA) has been used as photosensitizers for a wide range of malignant tumors as well as non-malignant disease. A new photosensitizer, 9-Hydroxypheophorbide-a (9-HpbD-a) was derived from Spirulina platensis. The aim of this study was to evaluate the anticancer effect and mechanism of photodynamic therapy in vitro using 9-HpbD-a and 670 nm diode laser on a HT-3 cervical cancer cell line. METHODS: We studied the cytotoxic effect of 9-HpbD-a and 670 nm diode laser in HT-3 cervical cancer cell line. The cultured HT-3 cells were treated with serial concentrations of 9-HpbD-a followed by various irradiation time (0, 5, 15, 30 min) and by various interval times (0, 3, 6, 9, 12 24 hours) until laser irradiation, then 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay was applied to measure the relative inhibitory effect of PDT. RESULTS: Optimal condition for laser irradiation time was 15 minutes and the cytotoxic effect according to the incubation time after 9-HpbD-a treatment was increased until 6 hours. Under confocal microscopy, to observe intracellular localization of 9-HpbD-a, cells dual-stained with 9-HpbD-a and panel of organelle-specific fluorescence probes (MitoTracker, LysoTracker, ER-Tracker), endoplasmic reticulum (ER) as a major accumulation organelle site in HT-3 cervical cell line for this new photosensitizer. CONCLUSION: The anticancer effect of PDT using 9-HpbD-a is effective in HT-3 cervical cancer cell line. This agent therefore represents a promising photosensitizing prodrug for the treatment of cervical cancer with PDT in clinical setting for the future.

ROS Formation and the Mechanism of Photodynamic Therapy Using 9-Hydroxypheophorbide-a on AMC-HN3 Human Head and Neck Carcinoma Cell Line / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: During PDT, photosensitizer accumulates in the cell and irradiation forms ROS. ROS leads to activation of apoptoticpathway and cell death. Elevated intracellular calcium is known to play important role in apoptotic pathway. There are two type of ROS formation. The type of ROS formation differs in type of photosensitizers. We designed the experiment to define the relationship of ROS and cell death in PDT. MATERIALS AND METHOD: AMC-HN3 cells were cultured. Using a CaspACE assay kit, we measured caspases-3 activity after PDT. We also observed intra-cellular calcium concentrations using confocal microscopy (calcium green-1 stain) after PDT. To determine which type of reaction occursduring ROS formation, MTT assay was performed. RESULTS: Confocal microscopy showed that ROS had formed at the site of photosensitizer formation after PDT. After PDT, intracellular calcium increased. MTT assay showed more viability increase in blocking type II reaction. Caspase assay showed highest level after 4hrs. CONCLUSION: ROS is formed at the site photosentizer formation after PDT. Type II reaction was the main type of ROS formation. Apoptosis was main pathway of cell death in low dose of photosensitizer after PDT.