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Photosensitizer effect of 9-hydroxypheophorbide α on diode laser-irradiated laryngeal cancer cells: Oxidative stress-directed cell death and migration suppression

View Article: PubMed Central - PubMed

ABSTRACT

The present study aimed to investigate the effect, and elucidate the potential mechanisms, of 9-hydroxypheophorbide α-based photodynamic therapy (9-HPbD-PDT) on apoptosis and necrosis induction, and migration suppression of laryngeal cancer AMC-HN-3 (HN-3) cells. Phototoxicity initiated by 9-HPbD-PDT on HN-3 cells was observed in a photosensitizer dose-dependent pattern. There was an initial increase of apoptotic cells coupled with gradual enhancement of reactive oxygen series (ROS) generation at lower doses of 9-HPbD. By contrast, at a higher dose of 9-HPbD, there was a clear increase of necrotic cells with a gradual decrease of ROS generation. Following PDT, an elevated percentage of apoptotic cells with shrinkage or condensing nuclei was observed using Hoechst 33342/propidium iodide double staining, and an upregulated expression of poly ADP-ribose polymerase was detected through western blotting. A disruption of the mitochondrial membrane potential was detected 2 h following PDT. Significant suppression of cell migration and downregulation of epidermal growth factor receptor (EGFR) expression were recorded following PDT. These results indicate that the distribution of photosensitizer leads to differences in the generation of ROS, which subsequently determines the type of cell death. Overall, mitochondrial activation under oxidative stress is important in the 9-HPbD-PDT-induced apoptosis of HN-3 cells. Migration suppression of HN-3 cells following PDT may be associated with the inhibited expression of EGFR, due to oxidative stress.

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9-hydroxypheophorbide α-PDT induced MMP collapse of laryngeal cancer AMC-HN-3 cells. MMP was detected 2 h following PDT using (A) flow cytometry and (B) confocal microscopy. (A) The distribution of MMP was shown as a blue-shaded area. Baseline MMP in the control group was labeled as a broken line. The spectral shift of the blue-shaded area to the left indicates mitochondrial membrane depolarization. A representative experiment of three repeats is presented. (B) Images of green fluorescence from rhodamine 123 stained mitochondria were captured by confocal microscopy (scale bar, 5 µm). PDT, photodynamic therapy; MMP, mitochondrial membrane potential.
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f3-ol-0-0-4889: 9-hydroxypheophorbide α-PDT induced MMP collapse of laryngeal cancer AMC-HN-3 cells. MMP was detected 2 h following PDT using (A) flow cytometry and (B) confocal microscopy. (A) The distribution of MMP was shown as a blue-shaded area. Baseline MMP in the control group was labeled as a broken line. The spectral shift of the blue-shaded area to the left indicates mitochondrial membrane depolarization. A representative experiment of three repeats is presented. (B) Images of green fluorescence from rhodamine 123 stained mitochondria were captured by confocal microscopy (scale bar, 5 µm). PDT, photodynamic therapy; MMP, mitochondrial membrane potential.

Mentions: Cells were stained with rhodamine 123, and MMP in single cells was monitored using flow cytometry and confocal microscopy. A collapsed MMP, shown as a leftward shift of the fluorescence curve from flow cytometry, was observed in a 9-HPbD dose-dependent manner (Fig. 3A). Polarized mitochondria were demonstrated by the presence of bright fluorescent spheres and tubes using confocal microscopy. A total of 2 h following PDT, the majority of the bright spheres became faint and rhodamine 123 fluorescence intensity was caliginous, indicating mitochondrial depolarization (Fig. 3B).


Photosensitizer effect of 9-hydroxypheophorbide α on diode laser-irradiated laryngeal cancer cells: Oxidative stress-directed cell death and migration suppression
9-hydroxypheophorbide α-PDT induced MMP collapse of laryngeal cancer AMC-HN-3 cells. MMP was detected 2 h following PDT using (A) flow cytometry and (B) confocal microscopy. (A) The distribution of MMP was shown as a blue-shaded area. Baseline MMP in the control group was labeled as a broken line. The spectral shift of the blue-shaded area to the left indicates mitochondrial membrane depolarization. A representative experiment of three repeats is presented. (B) Images of green fluorescence from rhodamine 123 stained mitochondria were captured by confocal microscopy (scale bar, 5 µm). PDT, photodynamic therapy; MMP, mitochondrial membrane potential.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997988&req=5

f3-ol-0-0-4889: 9-hydroxypheophorbide α-PDT induced MMP collapse of laryngeal cancer AMC-HN-3 cells. MMP was detected 2 h following PDT using (A) flow cytometry and (B) confocal microscopy. (A) The distribution of MMP was shown as a blue-shaded area. Baseline MMP in the control group was labeled as a broken line. The spectral shift of the blue-shaded area to the left indicates mitochondrial membrane depolarization. A representative experiment of three repeats is presented. (B) Images of green fluorescence from rhodamine 123 stained mitochondria were captured by confocal microscopy (scale bar, 5 µm). PDT, photodynamic therapy; MMP, mitochondrial membrane potential.
Mentions: Cells were stained with rhodamine 123, and MMP in single cells was monitored using flow cytometry and confocal microscopy. A collapsed MMP, shown as a leftward shift of the fluorescence curve from flow cytometry, was observed in a 9-HPbD dose-dependent manner (Fig. 3A). Polarized mitochondria were demonstrated by the presence of bright fluorescent spheres and tubes using confocal microscopy. A total of 2 h following PDT, the majority of the bright spheres became faint and rhodamine 123 fluorescence intensity was caliginous, indicating mitochondrial depolarization (Fig. 3B).

View Article: PubMed Central - PubMed

ABSTRACT

The present study aimed to investigate the effect, and elucidate the potential mechanisms, of 9-hydroxypheophorbide α-based photodynamic therapy (9-HPbD-PDT) on apoptosis and necrosis induction, and migration suppression of laryngeal cancer AMC-HN-3 (HN-3) cells. Phototoxicity initiated by 9-HPbD-PDT on HN-3 cells was observed in a photosensitizer dose-dependent pattern. There was an initial increase of apoptotic cells coupled with gradual enhancement of reactive oxygen series (ROS) generation at lower doses of 9-HPbD. By contrast, at a higher dose of 9-HPbD, there was a clear increase of necrotic cells with a gradual decrease of ROS generation. Following PDT, an elevated percentage of apoptotic cells with shrinkage or condensing nuclei was observed using Hoechst 33342/propidium iodide double staining, and an upregulated expression of poly ADP-ribose polymerase was detected through western blotting. A disruption of the mitochondrial membrane potential was detected 2 h following PDT. Significant suppression of cell migration and downregulation of epidermal growth factor receptor (EGFR) expression were recorded following PDT. These results indicate that the distribution of photosensitizer leads to differences in the generation of ROS, which subsequently determines the type of cell death. Overall, mitochondrial activation under oxidative stress is important in the 9-HPbD-PDT-induced apoptosis of HN-3 cells. Migration suppression of HN-3 cells following PDT may be associated with the inhibited expression of EGFR, due to oxidative stress.

No MeSH data available.


Related in: MedlinePlus