研究目的
To evaluate the cytotoxic effect of photodynamic therapy (PDT) combined with sulforaphane (SFN) on human head and neck cancer cells.
研究成果
Combination of PDT and SFN significantly enhanced cytotoxic effects on head and neck cancer cells by increasing ROS generation, which activated caspase pathways leading to apoptosis and necrosis. This suggests a potential for improved cancer therapy with reduced individual treatment intensities.
研究不足
The study is limited to in vitro experiments on a single cell line (AMC-HN3), which may not fully represent the complexity of human head and neck cancers in vivo. Clinical applicability and potential side effects were not assessed.
1：Experimental Design and Method Selection:
The study used a combination of PDT with SFN to enhance cytotoxicity on AMC-HN3 human head and neck cancer cells. Methods included MTT assay for cell viability, confocal microscopy for apoptosis/necrosis and ROS measurement, FACS for quantitative ROS analysis, and Western blot for caspase pathway activation.
2：Sample Selection and Data Sources:
The human head and neck cancer cell line AMC-HN3, derived from laryngeal carcinoma, was cultured in RPMI 1640 media with supplements.
3：List of Experimental Equipment and Materials:
Equipment included a confocal microscope (Zeiss LSM510 META), microplate reader (Bio-rad 550), FACS (FACSCalibur), incubator, and various chemicals such as Photofrin, SFN, MTT, H2DCFDA, antibodies for caspases and PARP.
4：Experimental Procedures and Operational Workflow:
Cells were treated with Photofrin and/or SFN, irradiated with a 630 nm diode laser for PDT, and assays were performed post-treatment. For ROS inhibition, sodium azide (SA) was used.
5：Data Analysis Methods:
Statistical analysis was done using SPSS 13.0, with t-test and Mann-Whitney U test for significance (p < 0.05).
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MTT
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenlyl-tetrazolium bromide
Sigma-Aldrich
Used in MTT assay to measure cell viability by detecting metabolic activity.
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DMSO
Dimethyl sulfoxide
Sigma-Aldrich
Solvent used to dissolve formazan crystals in MTT assay.
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Confocal microscope
Zeiss LSM510 META
Zeiss
Used for imaging apoptosis, necrosis, and ROS generation in cells via staining.
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Sodium azide
SA
Sigma-Aldrich
Scavenger of reactive oxygen species, used to inhibit ROS and study its role in cell death.
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Photofrin
Por?mer sodium
QTL Photo Therapuetics Inc.
Used as a photosensitizer in photodynamic therapy to generate reactive oxygen species upon light irradiation.
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RPMI 1640 media
HyClone
Cell culture medium for growing AMC-HN3 cancer cells.
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Fetal bovine serum
Gibco, BRL
Supplement for cell culture media to support cell growth.
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Streptomycin/penicillin
Gibco, BRL
Antibiotic supplement to prevent bacterial contamination in cell culture.
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Incubator
Maintains controlled environment for cell culture (5% CO2, 37°C).
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Diode laser
630 nm
Used for irradiating cells in photodynamic therapy to activate photosensitizer.
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Microplate reader
Bio-rad 550
Bio-rad
Measures absorbance at 540 nm for MTT assay to quantify cell viability.
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Hoechst 33342 dye
Sigma-Aldrich
Stains nuclei for apoptosis detection; apoptotic cells show bright blue condensation.
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Propidium iodide dye
Sigma-Aldrich
Stains nuclei for necrosis detection; necrotic cells show red staining.
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H2DCFDA
2’,7’-dichlorodihydro?uorescein diacetate
Molecular Probes
Fluorescent probe for measuring intracellular reactive oxygen species generation.
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FACS
FACSCalibur
Becton Dickinson
Quantitative analysis of ROS generation using fluorescence-activated cell sorting.
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Western blot equipment
Various (e.g., Bio-rad for PVDF membranes, Kodak for image analyzer)
Used to detect expression of caspase proteins and PARP in apoptosis pathways.
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Bradford assay reagent
Bio-rad
Used to determine protein concentration in samples for Western blot.
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