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Activation of two caspase cascades, caspase 8/3/6 and caspase 9/3/6, during photodynamic therapy using a novel photosensitizer, ATX-S10(Na), in normal human keratinocytes

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Abstract

Background/purpose

Photodynamic therapy (PDT) is a potent treatment for skin tumors. Although the therapeutic effect of PDT is supposed to be due to cellular cytotoxicity, the precise mechanism is still unknown. ATX-S10(Na) [13,17-bis(1-carboxypropionyl)carbamoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt], a novel hydrophilic chlorin photosensitizer, shows good accumulation in tumors and is suitable for use in PDT. In this study, we investigated the mechanism of PDT-induced cell death using ATX-S10(Na).

Methods

Following ATX-S10(Na) treatment for 12 h, normal human keratinocytes (NHK) were irradiated using a diode laser. PDT-induced cell death and the activity of various caspases were measured. Activation of Fas antigen was also determined by immunoprecipitation analysis. The expression of Bax, cytochrome c, and apoptosis-inducing factor (AIF) was determined by Western blotting.

Results

ATX-S10(Na)-PDT had induced apoptosis of NHK by 2 h and the maximal effect was observed at 6 h following irradiation. The effect was suppressed by pretreatment of NHK with inhibitors of caspases 3, 6, 8 and 9. A caspase activity assay revealed the sequential activation of caspases 8, 3 and 6, and caspases 9, 3 and 6, respectively. Immunoprecipitation analysis indicated multimerization of Fas antigen without Fas ligand binding in ATX-S10(Na)-PDT-treated NHK. Western blotting revealed cytosolic release of cytochrome c and AIF accompanied by decreased Bax expression in the cytosol.

Conclusions

ATX-S10(Na)-PDT induces apoptosis of NHK, and this was mediated by sequential activation of two caspase cascades, caspases 8, 3 and 6, and caspases 9, 3 and 6. This was accompanied by multimerization of Fas antigen and cytosolic release of cytochrome c and AIF.

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Acknowledgements

This study was supported in part by grants 1367086(HI), 10470184(AI-Y), and 11877138(AI-Y) from the Ministry of Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare, Japan. The technical assistance of Mrs. K. Nishikura and Miss M Nishinome, and the secretarial assistance of Miss M. Takahashi, are greatly appreciated.

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Authors and Affiliations

  1. Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaoka higashi, 078-8510, Asahikawa, Japan

    Hidetoshi Takahashi, Yasuhiro Itoh, Yuki Miyauchi, Akemi Ishida-Yamamoto & Hajime Iizuka

  2. Health Care Administration Center, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, 080-8555, Obihiro, Japan

    Susumu Nakajima

  3. Photochemical Co. Ltd., 5319-1 Haga, 701-1221, Okayama, Japan

    Isao Sakata

Authors
  1. Hidetoshi Takahashi
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  2. Yasuhiro Itoh
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  3. Yuki Miyauchi
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  4. Susumu Nakajima
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  5. Isao Sakata
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  6. Akemi Ishida-Yamamoto
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  7. Hajime Iizuka
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Correspondence to Hidetoshi Takahashi.

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Takahashi, H., Itoh, Y., Miyauchi, Y. et al. Activation of two caspase cascades, caspase 8/3/6 and caspase 9/3/6, during photodynamic therapy using a novel photosensitizer, ATX-S10(Na), in normal human keratinocytes. Arch Dermatol Res 295, 242–248 (2003). https://doi.org/10.1007/s00403-003-0424-5

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  • DOI: https://doi.org/10.1007/s00403-003-0424-5

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