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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-HPbD-PDT induced the cleavage of PARP (89 kDa), and inhibited the expression of EGFR (170 kDa). Laryngeal cancer AMC-HN-3 cells were treated with a sublethal dose of PDT, collected 24 h later, and subjected to western blot analysis. GAPDH (37 kDa) was used as a control. (A) Lane 1, control (no treatment); lane 2, 0.29 µg/ml 9-HPbD-PDT; lane 3, 0.59 µg/ml 9-HPbD-PDT. (B) Cells were treated with 0.59 µg/ml 9-HPbD-PDT alone or pretreated with 5 mM GSH or 2.5 mM ascorbic acid. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, poly ADP-ribose polymerase; EGFR, epidermal growth factor receptor; 9-HpbD, 9-hydroxypheophorbide α; PDT, photodynamic therapy; GSH, glutathione.
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f5-ol-0-0-4889: 9-HPbD-PDT induced the cleavage of PARP (89 kDa), and inhibited the expression of EGFR (170 kDa). Laryngeal cancer AMC-HN-3 cells were treated with a sublethal dose of PDT, collected 24 h later, and subjected to western blot analysis. GAPDH (37 kDa) was used as a control. (A) Lane 1, control (no treatment); lane 2, 0.29 µg/ml 9-HPbD-PDT; lane 3, 0.59 µg/ml 9-HPbD-PDT. (B) Cells were treated with 0.59 µg/ml 9-HPbD-PDT alone or pretreated with 5 mM GSH or 2.5 mM ascorbic acid. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, poly ADP-ribose polymerase; EGFR, epidermal growth factor receptor; 9-HpbD, 9-hydroxypheophorbide α; PDT, photodynamic therapy; GSH, glutathione.

Mentions: In contrast to the untreated group, there was clear inhibition of EGFR expression in PDT groups in a sensitizer dose-dependent manner (Fig. 5A). Downregulation in the expression of EGFR was partially inhibited when the cells were pretreated with ascorbic acid (Fig. 5B). As the native substrate of caspase-3, PARP was measured using western blotting. Similarly to EGFR expression, there was an elevated expression of PARP in a sensitizer dose-dependent manner (Fig. 5A). When cells were pretreated with GSH and ascorbic acid, upregulation of PARP was significantly inhibited (Fig. 5B).


Photosensitizer effect of 9-hydroxypheophorbide α on diode laser-irradiated laryngeal cancer cells: Oxidative stress-directed cell death and migration suppression
9-HPbD-PDT induced the cleavage of PARP (89 kDa), and inhibited the expression of EGFR (170 kDa). Laryngeal cancer AMC-HN-3 cells were treated with a sublethal dose of PDT, collected 24 h later, and subjected to western blot analysis. GAPDH (37 kDa) was used as a control. (A) Lane 1, control (no treatment); lane 2, 0.29 µg/ml 9-HPbD-PDT; lane 3, 0.59 µg/ml 9-HPbD-PDT. (B) Cells were treated with 0.59 µg/ml 9-HPbD-PDT alone or pretreated with 5 mM GSH or 2.5 mM ascorbic acid. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, poly ADP-ribose polymerase; EGFR, epidermal growth factor receptor; 9-HpbD, 9-hydroxypheophorbide α; PDT, photodynamic therapy; GSH, glutathione.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-ol-0-0-4889: 9-HPbD-PDT induced the cleavage of PARP (89 kDa), and inhibited the expression of EGFR (170 kDa). Laryngeal cancer AMC-HN-3 cells were treated with a sublethal dose of PDT, collected 24 h later, and subjected to western blot analysis. GAPDH (37 kDa) was used as a control. (A) Lane 1, control (no treatment); lane 2, 0.29 µg/ml 9-HPbD-PDT; lane 3, 0.59 µg/ml 9-HPbD-PDT. (B) Cells were treated with 0.59 µg/ml 9-HPbD-PDT alone or pretreated with 5 mM GSH or 2.5 mM ascorbic acid. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, poly ADP-ribose polymerase; EGFR, epidermal growth factor receptor; 9-HpbD, 9-hydroxypheophorbide α; PDT, photodynamic therapy; GSH, glutathione.
Mentions: In contrast to the untreated group, there was clear inhibition of EGFR expression in PDT groups in a sensitizer dose-dependent manner (Fig. 5A). Downregulation in the expression of EGFR was partially inhibited when the cells were pretreated with ascorbic acid (Fig. 5B). As the native substrate of caspase-3, PARP was measured using western blotting. Similarly to EGFR expression, there was an elevated expression of PARP in a sensitizer dose-dependent manner (Fig. 5A). When cells were pretreated with GSH and ascorbic acid, upregulation of PARP was significantly inhibited (Fig. 5B).

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