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Photodynamic therapy (PDT) is a treatment for the selective destruction of cancerous and non-neoplastic cells that involves the simultaneous presence of light, oxygen and a light-activatable chemical known as a photosensitizer. Curcumin is one of the most extensively investigated phytochemicals with chemopreventive potential and antitumor effects. In this study, the effect of a combination of PDT and curcumin on apoptotic cell death in AMC-HN3 cells and the molecular mechanism underlying apoptosis was examined to confirm the interaction between photofrin-induced PDT and curcumin during combined mortality. The combination treatment with curcumin and PDT inhibited approximately 70% of the cell viability after PDT, whereas the PDT and curcumin only groups showed a 50 and 10% decrease in cell viability, respectively. In addition, the combination treatment increased the apoptotic events, such as nuclear fragmentation and nuclear condensation. This combination group showed an increase in ROS generation that was higher than that observed after each single treatment. Compared to the single agent treatments, the combination therapy induced the enhanced loss of Δψm. Furthermore, the cytosolic levels of cytochrome
Photodynamic therapy (PDT) is a treatment for the selective destruction of cancerous and non-neoplastic cells that involves the simultaneous presence of light, oxygen and a light-activatable chemical known as a photosensitizer, with a photosensitive molecule that can be localized in the target cells (
The photosensitizer transfers energy from light to molecular oxygen to generate reactive oxygen species (ROS), particularly singlet oxygen, hydroxyl radicals and peroxides. This photosensitizer can react with oxygen via energy transfer processes, generating singlet oxygen (1O2) or it can participate in electron transfer processes, leading to radical formation (
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), also known as diferuloymethane, is the major yellow pigment in turmeric (
Conventional cancer therapies, including PDT and chemotherapy as a single modality, have a limited but important role in the overall treatment of most solid tumors. Therefore, the strategies of cancer treatment using combined therapies are considered more promising for higher efficacy, resulting in better survival rates. Cancer therapies combining chemoprevention including curcumin may have enhanced antitumor activity with a decrease in the toxicity caused by PDT. Recent evidence suggests that the photosensitizer effect of curcumin on UVB-irradiated HaCaT cells occurs through activation of the caspase pathways (
In this study, to confirm the interaction between photofrin-induced PDT and curcumin during combined mortality, this study examined effect and potential mechanism of a combination of PDT and curcumin on apoptotic cell death in AMC-HN3 cells.
Photofrin, a derivative of the haematoporphyrin, was purchased from LitePharm Tech, and was stable in PBS at −20°C in the dark. Curcumin (Sigma, St. Louis, MO, USA) was stable in DMSO (Sigma). A total of 100 mM of a stock solution of curcumin was stored at −20°C in the dark. The following antibodies were used: anti-caspase-8 (Calbiochem, La Jolla, CA, USA), anti-caspase-9 (Cell Signaling Technology, MA, USA), anti-capase-3 (Calbiochem), anti-cytochrome
The human head and neck cancer cell line (AMC-HN3) was kindly provided by Asan Medical Center (Seoul, Korea). The AMC-HN3 cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 100
The cells were seeded in 6-well plates, 96-well plates or plates, 100 mm in diameter. The cells were treated with a series of 2-fold dilutions of photofrin, starting at 50
The MTT assay was used to assess the cell viability of AMC-HN3 cells after the combination treatment. The cells attached in a 96-well plate (1,000 cells/well) were treated with 25
Hoechst 33342 (Sigma) and PI (Sigma) double staining was used to identify the cell death pattern. The nuclear morphology was assessed with the cell membrane-permeant supravital DNA dye Hoechst 33342 (excitation wavelength, 348 nm; emission, 479 nm). Hoechst 33342, unlike PI, enters and stains the nucleus of both viable cells and cells with apoptosis or necrosis. The plasma membrane integrity was assessed using the cell membrane-impermeant DNA dye PI (excitation wavelength 535 nm; emission 617 nm). Necrosis was determined based on the positive PI staining in red color, which is indicative of a loss of membrane integrity (
The intracellular accumulation of ROS was determined using H2DCFDA (2′,7′-Dichlorodihydro fluorescein diacetate, Molecular Probes, Eugene, OR, USA), as previously described (
Rhodamine 123 (Molecular Probes) was used to evaluate the mitochondrial membrane potential (Δψm), as predicted previously (
Six hours after PDT, the cells were washed twice with cold DPBS, and the cytosolic fraction and total protein were extracted in CE1 buffer (Qiagen, Valencia, CA, USA) and RIPA buffer, respectively. A Bradford assay (Bio-Rad) was used to determine the protein concentration by measuring the optical density at 595 nm using a spectrophotometer (Biochrom, Cambridge, UK). The protein samples were mixed with a 5X loading buffer (250 mM Tris, pH 6.8, 40% glycerol, 4% β-mercaptoethanol, 0.08% bromophenol blue, 8% sodium dodecyl sulfate), heat-denatured at 95°C for 10 min, loaded onto the 10% sodium dodecyl sulfate polyacrylamide gel and at 100 V for 90 min. After electrophoresis, gels with the resolved proteins were transferred to PVDF membranes (Bio-Rad) and blocked for 1 h in 10% skim milk. Each primary antibody (caspase-3, -9, cyto-chrome
The attached cells were co-treated with 25
The significance of the differences was evaluated using a Student’s t-test. A p-value <0.05 was considered significant.
A MTT assay was used to measure the cytotoxicity 24 h after PDT to assess the combination effect of curcumin and photofrin-induced PDT on AMC-HN3 cells. As shown in
Hoechst 33342 and PI double staining were performed to determine if the combination treatment induced enhanced apoptosis. There were only infrequent apoptotic bodies in curcumin only and PDT only groups. In contrast, more condensed/fragmented blue and pink nuclei as well as some pink intact nuclei were observed in the combination group. (
To determine if a pretreatment with curcumin affects the generation of ROS by PDT, the intracellular ROS level was detected using the fluorescent probe H2DCFDA, which is readily oxidized to 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. Compared to the control group, curcumin and PDT alone induced remarkable generation of ROS. The ROS signal induced by the combination group was higher than that of each single treatment (
To further examine the activation of mitochondria, the collapse of Δψm was quantified by flow cytometry. Compared to the control group, a decrease in Δψm, a leftward shift in the fluorescence curve, was clearly observed in the curcumin or PDT treatment alone group. The combination group showed a more intense loss of Δψm than that each single treatment group (
The collapse of Δψm by PDT has been suggested to be a key factor in the release of cytochrome
Mitochondrial release of cytochrome
In
The mitochondria-related apoptotic signals were investigated to further confirm the molecular mechanisms by which glutathione prevents apoptosis induced by the combination treatment. The collapse of Δψm in the combination therapy group was protected by a concomitant treatment with glutathione (
PDT must provide an enhanced therapeutic response to be used as a first-line curative modality (
The strategies of cancer treatment using combined therapies or combined agents with distinct molecular mechanisms are considered to be more promising for higher efficacy in the induction of apoptosis. The number of publications regarding the potentiated antitumor effects of cancer therapies using the chemopreventive agent, curcumin, has increased dramatically. In addition, common cancer therapies combined with these dietary compounds may exert enhanced antitumor activity through synergistic action (
Curcumin can both stimulate and inhibit apoptotic signaling. For example, curcumin induces apoptosis in human melanoma cells (30–60
The mitochondria are a major source of ROS in cells. As reported, ROS play an important role in controlling a range of cell functions, such as proliferation and apoptosis (
Loss of the mitochondrial membrane potential (Δψm) is associated with a dysfunction of the mitochondria, which can be detected in apoptotic cell death (
Caspases play critical roles in the initiation and execution of apoptosis and are activated by cytochrome
Hydroxyl radical and singlet oxygen have been reported to be important cellular mediators for PDT-induced responses (
In summary, these results suggest that the combined PDT with curcumin has enhanced cytotoxic and apoptotic effects on AMC-HN3 cells via a mitochondria-dependent apoptosis pathway. In addition, the generation of ROS plays an important role in this combination therapy-induced apoptosis.
We would like to thank the Medical Laser Research Center, Dankook University for the financial support.
Curcumin combined with PDT induces cell proliferation and apoptosis. (A) The cells were treated with 25
Curcumin combined with PDT changed mitochondria-induced ROS generation, collapse of MMP and cytochrome
Curcumin combined with PDT activated caspase-9, -3 and PARP. The total proteins (80
ROS generation and cytotoxicity induced by curcumin/PDT and the inhibitive effect of glutathione. (A) The cells were incubated with curcumin and glutathione for 6 h, treated with photofrin and glutathione for 6 h and irradiated with a laser without glutathione. The levels of ROS were measured using confocal microscopy. Magnification, x100. (B) The cells were incubated with curcumin and glutathione and then treated with photofrin and glutathione. The treated cells were irradiated with a 630-nm laser for 15 min without glutathione. After treatment, the cell viability was determined using the MTT assay. Each value represents the mean ± SD of three independent experiments.
Effect of the singlet oxygen-specific inhibitor glutathione on the combination treatment-induced MMP collapse, cytochrome