Taku Nakayama1, Naoko Nozawa2, Chiaki Kawada3, Shinkuro Yamamoto4, Takuya Ishii5, Masahiro Ishizuka6, Tsutomu Namikawa7, Shun-Ichiro Ogura8, Kazuhiro Hanazaki9, Keiji Inoue10, Takashi Karashima11. 1. Center for Photodynamic Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan; School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8501, Japan. Electronic address: taku.nakayama@kochi-u.ac.jp. 2. SBI Pharmaceuticals Co., Ltd., 1-6-1 Roppongi, Minato-ku, Tokyo, 106-6020, Japan. Electronic address: nnozawa@sbigroup.co.jp. 3. Department of Urology, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan. Electronic address: jm-kawadac@kochi-u.ac.jp. 4. Department of Urology, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan. Electronic address: jm-yamamoto-shinkuro@kochi-u.ac.jp. 5. SBI Pharmaceuticals Co., Ltd., 1-6-1 Roppongi, Minato-ku, Tokyo, 106-6020, Japan. Electronic address: taishii@sbigroup.co.jp. 6. SBI Pharmaceuticals Co., Ltd., 1-6-1 Roppongi, Minato-ku, Tokyo, 106-6020, Japan. Electronic address: mishizuk@sbigroup.co.jp. 7. Center for Photodynamic Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan; Department of Surgery I, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan. Electronic address: tsutomun@kochi-u.ac.jp. 8. Center for Photodynamic Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan; School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8501, Japan. Electronic address: ogura.s.ab@m.titech.ac.jp. 9. Center for Photodynamic Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan; Department of Surgery I, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan. Electronic address: hanazaki@kochi-u.ac.jp. 10. Center for Photodynamic Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan; Department of Urology, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan. Electronic address: keiji@kochi-u.ac.jp. 11. Department of Urology, Kochi Medical School, Kohasu, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan. Electronic address: karasima@kochi-u.ac.jp.
Abstract
BACKGROUND: Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to control the production of the intracellular photosensitizer protoporphyrin IX (PpIX) are commonly used clinically. Previously, we demonstrated that dormant and drug-induced dormancy-like cancer cells accumulated high PpIX levels, making them sensitive to ALA-PDT. Because EAU Guidelines awarded a level of evidence of 1a to mitomycin C, the drug is widely used to treat bladder cancer. In this study, we investigated that the effect of mitomycin C-induced cell cycle arrest on porphyrin metabolism, including that induced by ALA-PDT. METHODS: T24 human urinary bladder carcinoma cells were selected for this research. T24 cells were irradiated using a light-emitting diode emitting red light for the ALA-PDT assay. Cell cycle analysis was conducted by flow cytometry using bromodeoxyuridine. Cell viability was confirmed using the MTT or colony formation assay. Furthermore, mRNA gene expression analysis was performed using our previously reported methods. RESULTS: The cell cycle of T24 cells was arrested at G2/M phase by mitomycin C. PpIX accumulation was dramatically increased by mitomycin C treatment. Cell viability after ALA-PDT was remarkably decreased by mitomycin C pretreatment. The gene expression of porphyrin transporters was consistent with the metabolic and morphological results. Finally, we confirmed that ALA-PDT combined with mitomycin C treatment exerted a long-term inhibitory effect on cell proliferation. CONCLUSION: This study demonstrated a new approach to enhance the effects of ALA-PDT using drugs that induce a dormancy-like status and upregulate porphyrin metabolism.
BACKGROUND: Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to control the production of the intracellular photosensitizer protoporphyrin IX (PpIX) are commonly used clinically. Previously, we demonstrated that dormant and drug-induced dormancy-like cancer cells accumulated high PpIX levels, making them sensitive to ALA-PDT. Because EAU Guidelines awarded a level of evidence of 1a to mitomycin C, the drug is widely used to treat bladder cancer. In this study, we investigated that the effect of mitomycin C-induced cell cycle arrest on porphyrin metabolism, including that induced by ALA-PDT. METHODS: T24 humanurinary bladder carcinoma cells were selected for this research. T24 cells were irradiated using a light-emitting diode emitting red light for the ALA-PDT assay. Cell cycle analysis was conducted by flow cytometry using bromodeoxyuridine. Cell viability was confirmed using the MTT or colony formation assay. Furthermore, mRNA gene expression analysis was performed using our previously reported methods. RESULTS: The cell cycle of T24 cells was arrested at G2/M phase by mitomycin C. PpIX accumulation was dramatically increased by mitomycin C treatment. Cell viability after ALA-PDT was remarkably decreased by mitomycin C pretreatment. The gene expression of porphyrin transporters was consistent with the metabolic and morphological results. Finally, we confirmed that ALA-PDT combined with mitomycin C treatment exerted a long-term inhibitory effect on cell proliferation. CONCLUSION: This study demonstrated a new approach to enhance the effects of ALA-PDT using drugs that induce a dormancy-like status and upregulate porphyrin metabolism.