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We investigated cellular responses to chlorin-based photosensitizer DH-II-24 under darkness in human gastric adenocarcinoma AGS cells. Cells were loaded with 0.5–10 μg/mL DH-II-24 for 12 h, and intracellular reactive oxygen species (ROS) and intracellular Ca2+ levels, in situ tissue transglutaminase (tTGase) activity, cell viability, cell morphology and cell cycle were examined. DH-II-24 treatment had no effect on intracellular ROS production or cell morphology, and did not induce cell detachment at any concentrations tested. In addition, cell viability and cell cycle progression were not altered by the photosensitizer. However, DH-II-24 treatment elevated the basal level of intracellular Ca2+ in a dose-dependent manner and inhibited tTGase activity without affecting tTGase expression levels. Furthermore, DH-II-24 inhibited lysophosphatidic acid-induced activation of tTGase in a dose-dependent manner. In contrast, photodynamic therapy (PDT) with 1 μg/mL DH-II-24 significantly elevated intracellular ROS and in situ tTGase activity in parallel with a rapid and large increase in intracellular Ca2+ levels. DH-II-24-mediated PDT decreased cell viability and induced cell detachment. These results demonstrate that DH-II-24 treatment alone under darkness induced different cellular responses to DH-II-24-mediated PDT. (Cancer Sci 2011; 102: 549–556)
Photodynamic therapy (PDT) is a promising therapeutic modality for cancer treatment that involves photosensitizers, irradiation with appropriate wavelengths, and molecular oxygen.(1,2) Activated photosensitizers lead to significantly increased levels of intracellular reactive oxygen species (ROS) and intracellular Ca2+. These changes result in the killing of tumor cells either directly or by damaging the tumor-associated vasculature through apoptosis and necrosis. In addition, PDT indirectly induces a significant stimulatory effect on the immune system.(3–6)
Photodynamic therapy has advantages over other methods of cancer treatment, such as non-invasive treatment, by avoiding systemic immune suppression and selectively destroying tumors.(7–13) However, it is essential to consider the side-effects of non-irradiated photosensitizers under darkness in patients. Photofrin, the most widely used photosensitizer in clinical PDT, has several weaknesses within clinical applications, including adverse reactions involving skin damage caused by slow clearance from the circulation system and low photosensitivity.(2,3,14) To avoid adverse effects, patients should remain in a dark condition until excretion of the photosensitizer following intravenous administration. Remaining in the dark reduces quality of life.(15,16) New photosensitizers have been developed with reduced side-effects and increased effectiveness.(2,8,14,17,18) Previously, we reported a new chlorin-based photosensitizer, DH-II-24, and examined its photosensitivity in human gastric adenocarcinoma (AGS) cells, human colorectal carcinoma (HCT116) cells and xenografts.(19,20) Photo-activated DH-II-24 elevated the levels of intracellular ROS and Ca2+, indicating that DH-II-24 is effective for use in PDT.(19) Several reports focus on the PDT-induced mechanisms of cell death using photosensitizers.(19,21,22) However, the responses of cells or animals to non-irradiated photosensitizers remains to be elucidated.
Dark toxicity of photosensitizers has been studied through cell viability assays in various cell types.(23–28) Incubation with various concentrations (up to 6 μM) of the valine-derivatized porphyrin for 24 h showed little or no toxicity under darkness in human breast carcinoma (MCF-7) cells.(25) Meta-tetra(hydroxyphenyl) chlorin (mTHPC; Foscan) was applied to several cancer cell lines, including gall bladder cancer cells, bile duct cancer cells and MCF-7 cells,(23,28) and cytotoxicity was examined using methylthiazolyldiphenyl-tetrazoliumbromide (MTT) assays. In gall bladder and bile duct cancer cells, no dark toxicity was observed with 2–3 μg/mL mTHPC, although cell viability rapidly decreased in response to higher concentrations.(23) Similar results were obtained using mTHPC in MCF-7 cells.(28) However, much higher concentrations of mTHPC derivatives, such as a PEG2000 derivative of mTHPC (m-THPC MD) and a liposomal formulation of mTHPC (Foslip), were required to induce similar dark toxicity.(23,28) Dark toxicity was also investigated using other photosensitizers such as hematoporphyrin monomethyl ether,(24) 18m-ALA (a dendrimer containing 18 aminolevulinic acids)(26) and dithiaporphyrin(27) in various cancer cells. The results indicated minimal dark toxicity in concentration ranges similar to porphyrin and mTHPC. Thus, dark toxicity of various photosensitizers has been examined in cancer cells. However, the effects of photosensitizers on intracellular responses remain to be elucidated.
In the present study, we examined cellular responses to non-irradiated DH-II-24 in human gastric adenocarcinoma AGS cells, including intracellular ROS and Ca2+ accumulation, in situ tissue transglutaminase (tTGase) activity, cell viability and cell cycle progression. tTGase, a multifunctional protein, is regulated by several factors including intracellular Ca2+ and redox potential, and is involved in a wide variety of cellular processes including cell death, inflammation and wound healing.(29) It is reported that PDT with DH-II-24 elevated the level of intracellular Ca2+ and tTGase is activated by intracellular Ca2+ in AGS cells.(19,30) DH-II-24 treatment had no significant effect on intracellular ROS levels, but the basal level of intracellular Ca2+ was elevated and in situ tTGase activation was inhibited. However, PDT with DH-II-24 (1 μg/mL) treatment decreased cell viability in parallel with elevated intracellular ROS levels and in situ tTGase activity. In addition, dark toxicity was not observed at up to 10 μg/mL DH-II-24, a 10-fold greater concentration than that used for PDT. Therefore, higher-concentration DH-II-24 treatments may be more beneficial for cancer treatment by achieving more efficient PDT, while maintaining the previously known advantages of photo-activation with a long wavelength light, significant photoxicity and rapid clearance from the body.
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Although a tremendous number of photosensitizers have been reported for in vitro and in vivo PDT treatments, only a few of these photosensitizers have ideal properties for clinical trials, such as chemical purity, tumor-selective accumulation, activation at longer wavelengths of light for tissue penetration, rapid clearance from the body and high photosensitivity.(35–37) Thus, a new generation of photosensitizers are still under investigation. Photosensitizers can be structurally classified into porphyrin derivatives, chlorins, phthalocyanins and porphycenes, each reacting differently and having different photosensitivities.(38) Photosensitizers are activated by specific wavelengths of light and can increase intracellualar ROS and Ca2+ levels, important factors for PDT-induced cell death.(3–6,8,19,39,40) Thus, the determination of cell death mechanisms has been an important topic in PDT treatment. However, to improve PDT efficacy by minimizing side-effects, it is essential to understand the effects of photosensitizers accumulated in non-target tissues under darkness. Although there are several reports on dark toxicity of photosensitizers,(23–28) to our knowledge this is the first report focusing on cellular responses to photosensitizers under darkness. Furthermore, this report provides a foundation for understanding the possible side-effects of photosensitizers that remain in non-targeted tissues, including skin, after PDT treatment.
We previously reported that DH-II-24-mediated PDT increased the levels of intracellular ROS and Ca2+ in AGS cells,(19) and that TGase was activated by intracellular ROS and Ca2+ in fibroblasts.(30,32,41) Thus, we examined changes in intracellular ROS and Ca2+ levels, in situ tTGase activity and cell viability in response to DH-II-24 treatment in human gastric adenocarcinoma cells. DH-II-24 treatment without photo-activation had no effect on intracellular ROS levels and cell cycle progression, and was not cytotoxic. However, this treatment did elevate the basal level of intracellular Ca2+ and inhibited tTGase activity without altering tTGase expression levels. On the other hand, DH-II-24-mediated PDT elevated intracellular ROS levels and in situ tTGase activity in parallel with a transient but strong increase in intracellular Ca2+ resulting in decreased cell viability. The expression level of tTGase was not altered by PDT (data not shown). Thus, DH-II-24 treatment under darkness induced different cellular responses than DH-II-24-mediated PDT in AGS cells. Furthermore, DH-II-24 treatment did not induce dark toxicity at concentrations up to 10 μg/mL, 10-times greater than that used for PDT (1 μg/mL), indicating that more effective PDT can be performed using higher concentrations of DH-II-24.
It is interesting that DH-II-24 treatment inhibited tTGase activity, while DH-II-24-mediated PDT stimulated tTGase activity. It was expected that DH-II-24 would activate tTGase because TGase is activated by intracellular Ca2+.(29,30,41) However, tTGase activity was significantly inhibited by DH-II-24 treatment and tTGase stimulation by LPA was also prevented by the photosensitizer in a dose-dependent manner. In addition, we performed a tTGase activity assay in the presence of 20 μM aminoguanidine, a potent inhibitor of amineoxidase, and similar inhibitory effects of DH-II-24 on in situ tTGase activity were also observed (data not shown). DH-II-24 treatment increased the basal level of intracellular Ca2+ 9 h after incubation with 10 μg/mL of the photosensitizer with no effect on intracellular ROS levels. This indicated that elevation of basal intracellular Ca2+ levels was not enough to alter in situ tTGase activity. Thus, DH-II-24 inhibited tTGase in AGS cells through an unknown mechanism(s) independent of intracellular Ca2+ levels. It would be very useful to examine the inhibitory effects of other photosensitizers on tTGase activity and their mechanisms of tTGase inhibition.
Tissue transglutaminase is a multifunctional enzyme that is implicated in a number of cellular events including apoptosis and cell adhesion, mobility and invasion.(29) There is a report that PDT induced resistance to trypsinization and elevated in vitro tTGase activity in Met B cells;(42) however, in situ tTGase activation by PDT is not known. In this report, we demonstrated that DH-II-24-mediated PDT activated in situ tTGase and elevated the levels of intracellular ROS and Ca2+. Because tTGase is activated by intracellular ROS and Ca2+,(30,32,41) it is possible that DH-II-24-mediated PDT might activate tTGase via elevation of intracellular ROS and Ca2+ levels. Considering the role of tTGase in apoptosis,(29) it is likely that tTGase mediates cell death by PDT, although it is necessary to elucidate the mechanism(s) of tTGase activation by PDT and its role in PDT-induced cell death.
In summary, we examined cellular responses to a cholin-based photosensitizer, DH-II-24, under darkness in human gastric adenocarcinoma AGS cells. DH-II-24 without photo-activation was not cytotoxic and increased basal levels of intracellular Ca2+ and inhibited tTGase, whereas DH-II-24-mediated PDT was cytotoxic, elevated intracellular ROS levels, increased in situ tTGase activity and showed a transient peaking of intracellular Ca2+ levels.