Induction of cell death by Ta3N5 in the human cancer cell line
Description
Photocatalysts are widely used in modern society, and their applications extend to medicine. Until now, ultraviolet light irradiation has been required for photocatalysis, but tantalum nitride (Ta3N5) has attracted attention as a next-generation photocatalyst because it produces reactive oxygen species (ROS) upon visible light irradiation. However, it has never been used in life science, and its toxicity was unknown. In the present study, we evaluated the cytotoxicity of Ta3N5 using U2OS cells in terms of light dose, irradiation time, Ta3N5 dope, and light wavelength. We also investigated the mode and mechanism of induced cell death. The results showed that cytotoxicity increased in a dose-, time-, and Ta3N5 dope-dependent manner, especially with blue light irradiation. On the other hand, high-dose light irradiation induced cytotoxicity with or without Ta3N5, indicating that green light and red light irradiation, which can selectively toxicize Ta3N5-added cells, may also be effective. We also showed that Ta3N5 induces secondary necrotic cell death in U2OS cells. These data indicate that Ta3N5 may be a photocatalyst that can be used in the medical and life science fields.
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Light Irradiation Six LED panels capable of emitting blue (460-470 nm), green (520-525 nm), and red (620-625 nm) light were mounted on an aluminum plate. After wiring, the light intensity could be adjusted. To irradiate the sample evenly with light, a light irradiation device was fabricated with a stand and at a distance from the irradiated object. Cell Culture U2OS cells (human osteosarcoma cell line) were grown in Dulbecco's Modified Eagle Medium (DMEM (+/+)) supplemented with 10% fetal bovine serum (FBS) (NICHIREI, Tokyo, Japan) and 100 U/ml Penicillin-Streptomycin as antibiotics. Preparation of Ta3N5 suspension 0.5 g of Ta3N5 powder (Kozyundo Chemical Laboratories, Saitama, Japan) was added to 5 ml of phosphate-buffered saline PBS (-) to make a 0.1 g/ml Ta3N5 suspension, which was then autoclaved. Trypan blue stain The cells were seeded at 0.015 × 106 cells / well in 8 well chamber slides and incubated for 1 day. 0, 1.6, and 3.2 mg/ml of Ta3N5 was added to the green light-irradiated cell groups at a ratio of culture medium: Trypan Blue Solution (Thermo Fisher Scientific) = 1:1, stained, washed, and sealed. Measurement of viable cells in Ta3N5-added group U2OS cells were seeded at 0.03 × 106 cells/well in 48 well Plates. After 1 day of culture, Ta3N5 was added at 0, 1.6, and 3.2 mg/ml to each 16 well and irradiated with blue, green, and red light for 1, 2, and 4 hours. Cell viability was then measured by the WST-8 assay. Specifically, after washing with 100 μl of PBS (-), 100 μl of Recording Medium for measurement and 10 μl of Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) were added and incubated at 37°C for 3 hours. Then, 80 μl of the supernatant was transferred to a 96 well plate and measured at 450 nm absorbance using an Enspire 2300 absorbance meter (Perkinelmer, Massachusetts, USA). This is measured by absorbance at 450 nm to allow measurement of the number of viable cells. Identification of the cell death pathway of Ta3N5 by fluorescent immunostaining Fluorescent immunostaining was performed to evaluate the morphology of apoptotic cells. Cells were washed with PBS (-), stained with MEBCYTO Apoptosis Kit (Annexin V-FITC Kit, MBL, Nagoya, Japan). Detection of DNA fragmentation in dead cells U2OS cells were seeded in 6 mm Dish at 1.0 × 107 cells; after 24 h, cells were harvested using a cell scraper. DNA was then isolated and the DNA ladder assay was performed using the Apopladder Ex™ kit (Takara Biochemical, Shiga, Japan). Statistics Analysis All P values for each test were performed at 5%, with P < 0.05 indicating a significant difference. Cytotoxicity and nuclear size are all expressed as mean ± standard error. One-way ANOVA was used to compare three or more groups. When a significant difference was found, a multiple comparison method with one-way ANOVA Tukey's test was used to test the significance of the data here.