Cell death and intracellular distribution of hematoporphyrin in a KB cell line.
OBJECTIVE
The objective of this study is to investigate the effect of intracellular photosensitizer distribution on tumor cell death after photodynamic therapy (PDT).


BACKGROUND
The photosensitizer accumulates in tumor tissue during PDT, and generates intracellular reactive oxygen species (ROS), resulting in tumor cell death.


METHODS
This study was carried out to elucidate the effects of PDT in a KB oral cancer cell line using hematoporphyrin with irradiation at 635 nm and 5 mW/cm(2). After irradiation, the MTT reduction method, agarose gel electrophoresis, flow cytometry, and Diff-Quick staining were performed. The intracellular ROS level was measured by DCF-DA. Intracellular hematoporphyrin was monitored with a confocal microscope, and Western blot and caspase activity assays were performed.


RESULTS
In our study, cell survival was reduced by about 50% after 3 h of hematoporphyrin incubation time. In DNA fragmentation, flow cytometry, and Diff-Quick assay, necrosis was identified within 12 h and apoptosis soon thereafter. Confocal microscopy revealed that hematoporphyrin was localized in the cell membrane, cytoplasm, and nucleus as time passed. The quantities of intracellular ROS correlated with the time of hematoporphyrin accumulation. Additionally, Western blot analysis of Bcl-2/Bax, the release of cytochrome C, and activity of caspase-3 and caspase-9 showed that apoptosis followed the mitochondria-dependent pathway.


CONCLUSIONS
PDT with hematoporphyrin in the KB cell line showed morphological changes of cell necrosis and apoptosis, which were associated with the time of distribution and localization of hematoporphyrin. Also, the apoptosis evoked followed the mitochondria-dependent pathway.
Department of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University, Bug-Gu, Gwangju, Korea.
Hongran
H
Choi
Wonbong
W
Lim
Ji-Eun
JE
Kim
Inae
I
Kim
Jinan
J
Jeong
Youngjong
Y
Ko
Jongwoon
J
Song
Sunyeol
S
You
Doman
D
Kim
Misook
M
Kim
Byung-Kuk
BK
Kim
Okjoon
O
Kim
D017209:Apoptosis; D015153:Blotting, Western; D016923:Cell Death; D045744:Cell Line, Tumor; D002470:Cell Survival; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D006414:Hematoporphyrin Photoradiation; D006415:Hematoporphyrins / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D007624:KB Cells; D018613:Microscopy, Confocal; D009062:Mouth Neoplasms / Q000473:pathology*; D017319:Photosensitizing Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013194:Staining and Labeling
D006415:Hematoporphyrins; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species