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Detection of photodynamic therapy-induced early apoptosis in human salivary gland tumor cells in vitro and in a mouse tumor model
Oral Oncology (2004) 40 787–792
http://intl.elsevierhealth.com/journals/oron/
Detection of photodynamic therapy-induced early apoptosis in human salivary gland tumor cells in vitro and in a mouse tumor model Tadayoshi Kanekoa,*, Hiroshige Chibaa, Takashi Yasudaa, Kaoru Kusamab a
Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan b Deparment of Oral Pathology, Meikai University School of Dentistry, Sakado, Saitama, Japan Received 11 December 2003; accepted 31 January 2004
Available online KEYWORDS
Summary We studied the detection of apoptosis of malignant human salivary gland tumor cells induced by photodynamic therapy (PDT) using the photosensitizer monoL-aspartyl chlorin e6 (NPe6) in vitro and in vivo in mice receiving transplanted human salivary gland tumor (HSG) cells. An immunohistocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin (CK) 18 by activated caspase, was applied to detect the apoptosis of HSG cells induced by PDT. Significant amounts of immunoreactive products were observed in the cytoplasm of HSG cells after PDT. In vitro, M30positive cells increased from 2 h after PDT, increased rapidly from 8 h and reached a peak 24 h after PDT. In vivo, a peak of early apoptosis was confirmed two hours after PDT. In comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, the destroyed tumor cells were observed sporadically 24 h after PDT. These results suggest that immunohistocytochemical staining with the MoAb M30 may be useful for detecting early apoptosis induced by PDT. Futhermore, PDT using NPe6 is effective in inducing apoptosis of HSG cells at an early stage, which suggests the possibility of the therapy being ideal for treatment of human malignant neoplasms. c 2004 Published by Elsevier Ltd.
Photodynamic therapy; Human salivary gland tumor cells; M30
Introduction Photodynamic therapy (PDT) may result in either apoptotic or necrotic cell death.1 PDT may interact * Corresponding author. Tel.: +81-33342-6111, fax: +8133342-1723. E-mail address: [email protected] (T. Kaneko).
1368-8375/$ - see front matter c 2004 Published by Elsevier Ltd. doi:10.1016/j.oraloncology.2004.01.007
with many pathways but it is unclear exactly what is responsible for early apoptosis following PDT. Many stimuli may trigger apoptosis. These stimuli interact with mitochondria, which are thought to be key regulators of apoptosis by activations of the caspase cascade.2 The monoclonal antibody M30 reacts specifically with the product resulting from the cleavage of CK 18 by activated caspase3;4 and is
788 known to be useful for detection of early apoptosis in human salivary glands and tumors derived from salivary glands.4–8 We examined the early apoptosis-inducing activities of PDT in combination with NPe6,9 a relatively new photosensitizer, for HSG cells by the immunohistocytological method using MoAb M30 and compared the results with those obtained using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method.10
T. Kaneko et al.
In vitro study of photosensitizer properties Cell culture A human salivary gland tumor HSG cell line was maintained in monolayer cultures at 37 C in RPMI 1640 medium supplemented with 10% heatinactivated FBS in a humidified 5% CO2 atmosphere. Assay for cytotoxic and antitumor activities
Materials and methods Materials The following chemicals and reagents were obtained from the indicated companies: NPe6 (Meiji Seika, Ltd., Tokyo, Japan, structure shown in Fig. 1); diode laser (Matsushita, Ltd., Tokyo, Japan); RPMI 1640 medium (GIBCO, Grand Island, NY, USA); fetal bovine serum (FBS, GIBCO-BRL , Life Technologies Inc., Gaithersburg, MD, USA); WST-1, 1Methoxy PMS (WST, Dojin Chem. Ind., Kumamoto, Japan); MoAb M30 (Boehringer Mannheim, GmbH, Mannheim, Germany); MoAb DC-10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); streptavidin-peroxidase (GIBCO-BRL , Life Technologies Inc.); biotinylated horse anti-mouse Ig G (H + L) antibody (Vector Laboratories lnc., Burlingame, CA, USA); 3,30 -diaminebenzidine tetrahydrochloride (DAB, Sigma Chemical Industries., St. Louis, MO, USA); a TACS in situ apoptosis detection kit (Trevigen, Inc., Gaithersburg, MD, USA).
The relative viable cell number was determined by the WST method. Near-confluent HSG cells grown in 96-microwell plates (Falcon, flat bottom, treated polystyrene, Becton Dickinson and Company., Franklin Lakes, NJ, USA) were incubated for 24 h, and then were incubated for 2 more hours with NPe6 at 0, 1.25, 2.5, 5.0, 10, 20 and 40 lg/ml in RPMI 1640 medium supplemented with 10% FBS, washed once with phosphate-buffered saline (PBS, 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.4). PDT was performed with a diode laser (absorption peak: 664 nm, output: 100 mW/cm2 and total radiation: 10 J/cm2 ) and the materials were then incubated in the dark for 24 more hours after PDT in RPMI 1640 medium supplemented with 10% FBS, and then incubated for 4 h with 0.2 mg/ml WST. After the medium was removed, the cells were lysed with 100 ll DMSO and the relative viable cell number was determined by measuring the absorbance at 650 nm of the cell lysate using a model 550 microplate reader (Bio-Rad laboratories, Inc., Hercules, CA, USA) Detecting apoptosis induced by PDT
Figure 1 NPe6 was used as a photosensitizer. NPe6 has a ring of 4 pyrroles and asparatic acid combining with the D ring. The molecular weight is 799.7 and the absorption peak is 664 nm. NPe6 is a compound with a high affinity for tumor.
A chamber slide (Nalge Nunc International, Lab-Tek Brand Products., Naperville, IL, USA) with 5 · 104 /ml of HSG cells was incubated, then 2 h later 10 lg/ml of NPe6 was added, and PDT was performed under the same conditions as described above. The samples were allocated to experimental Group 1 (0.5 h after laser irradiation), Group 2 (at 1 h), Group 3 (at 2 h), Group 4 (at 4 h), Group 5 (at 8 h), Group 6 (at 24 h), Group 7 (at 48 h) and control group (no laser irradiation). Cultured cells on chamber slides were fixed at those times with a mixture of 95% ethanol and 5% acetic acid for 10 min at 4 C. Thereafter the samples were stained immunocytochemically. The mean numbers of M30-positive cells in five different fields under a microscope (5 · 3.3 times) in each group were calculated and were compared.
Apoptosis of HSG cells by PDT
Animal experiments Animals Male BALB/c-nude mice aged 4 weeks were used as experimental animals. All animals received good care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”11 and all experiments were carried out in accordance with protocols approved by the local Experimental Animal Research Committee.
789 (1:200) at room temperature for 30 min. After the samples were washed with PBS three times, the samples were color-developed with DAB solution and washed with PBS. After washing with water, the nuclei were stained with Mayer hematoxylin, washed with water, dehydrated and mounted. For the activation of antigenicity, the samples were treated beforehand with 0.01M-citrate buffer (pH 6.0) by microwave for 15 min.
Detection of apoptotic cells by DNA nick end labeling of tissue sections
Detecting apoptosis induced by PDT in vivo Cell suspensions of human salivary gland tumor cells (5 · 106 ) were injected aseptically into the subcutaneous tissues of the femur of male BALB/cnude mice (mean body weight: 25 g) aged 4 weeks. NPe6 (10 mg/kg) was injected intravenously into the tissues 2 weeks after transplantation of tumor cells, by which time the tumors had reached about 5 mm in diameter, and 2 h later tumor was irradiated with a diode laser (664 nm, 100 J/cm2 ). The mice were allocated to Group 1 (sacrificed at 0.5 h after laser irradiation), Group 2 (at 1 h), Group 3 (at 2 h), Group 4 (at 4 h), Group 5 (at 24 h), Group 6 (at 72 h) and control group (sacrificed without laser irradiation) and samples were collected from each group. These test samples were fixed in 10% buffered neutral formalin, embedded in paraffin and thin-sectioned in the routine manner. After deparaffinization, the thin-sections were reacted with methanol containing 0.3% H2 O2 at room temperature for 15 min to inhibit endogenous peroxidase. Thereafter the samples were stained immunohistochemically. The mean numbers of M30-positive cells in five different fields under a microscope (5 · 3.3 times) in each group were calculated and were compared. DNA fragmentation was also investigated in the in vivo group by the TUNEL method.
In order to demonstrate the apoptosis of cells induced by PDT, DNA fragmentation was visualized by the modified method using a TACS in situ apoptosis detection kit. Cells treated by PDT on slides were fixed with 10% neutralized buffered formalin for 10 min at room temperature and after washing with water, each slide was dried for 30 min at 56 C. The slides were rinsed with PBS and treated with 10 lg/ml proteinase K for 5 min at room temperature. To block endogenous peroxidase activity, the slides were immersed in 2% H2 O2 solution for 5 min at room temperature. After washing in PBS and pretreatment with labeling buffer, each slide was incubated with labeling mixture (TdT, biotin-dUTP) for 60 min at 37 C. After stopping the reaction, each slide was treated with 2% bovine serum albumin in PBS (BSA-PBS) for 15 min to block nonspecific reactions, and then incubated with diluted streptavidin-peroxidase (1:200). The slides were washed with PBS three times and immersed for 10 min in 0.05% 3,30 -diaminebenzidine tetrahydrochloride (DAB) in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% H2 O2 . Each slide was counterstained with Mayer’s hematoxylin and mounted.
Results and discussion Immunologic procedure After samples were washed with phosphate buffer solution (PBS), the samples were reacted with 2% bovine serum albumin BSA (bovine serum albumin)-PBS at room temperature for 15 min to block non-specific reaction and reacted with MoAb M30 (1:30) for 60 min. After the samples were washed with PBS three times, they were reacted with anti-mouse IgG (H + L) horse antibody (1:200) at room temperature for 30 min. After the samples were washed with PBS three times, the samples were reacted with streptavidin-peroxidase solution
We have previously shown that the distribution of CK 18 in the salivary glands and pleomorphic adenomas and extent of apoptosis in them were determined using a MoAb M30, which reacts with the caspase cleavage neo-epitope in CK 18 exposed during early apoptosis.6 We developed this to detect the apoptosis of HSG cells induced by PDT.7 To establish whether MoAb M30 could detect activity of the early apoptosis induced by PDT, we prepared an in vivo mouse model of transplanted HSG cells and irradiated the HSG cells both in vivo and in vitro.
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Figure 2 Number of viable HSG cells determined in the laser-irradiation groups () compared with the non-laserirradiation group () by the WST method. Each value represents a mean ± SD for triplicate assays.
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The antitumor activity by PDT against HSG cells was clearly obtained concentration-dependently in the range of 5–20 lg/ml of NPe6 in the irradiation groups as compared with the non-irradiation group. There were only 8.1% viable cells following PDT after treatment with 20 lg/ml of NPe6, thus marked antitumor activity was obtained (Fig. 2). Concerning detection of apoptosis induced by PDT in vitro, M30- positive cells increased in Group 3 from 2 h after PDT, increased rapidly in Group 5 from 8 h and reached a peak in Group 6 at 24 h. Thereafter the positive cells decreased markedly and were nearly the same in Group 7 at 48 h after PDT as those in Group 4 at 4 h. M30-positive cells decreased from 24 h after PDT (Figs. 3 and 4). In vivo, M30-positive cells increased markedly in Group 1 from 0.5 h after PDT and were distributed more entirely throughout tumor tissues than in the control group. The number of positive cells tended to further increase in Group 2 and peaked in Group 3. Positive cells were observed continuously in Group 5 until 24 h after PDT. Apoptosis was observed in the whole tumor area including the deep part of the tumor by 24 h after PDT. Thereafter M30-positive cells themselves decreased (Figs. 5 and 6). A tendency to delayed onset of apoptosis was observed in the deep part of the tumor compared to the superficial parts of the tumor. In the induction of apoptosis in vivo, TUNELpositive cells increased in the implanted tumor part in Group 5 from 24 h after PDT by the TUNEL
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Figure 3 Cytological apoptosis of HSG cells induced by PDT. M30-positive cells increased in Group 3 from 2 h after PDT, increased rapidly in Group 5 from 8 h and reached a peak in Group 6 at 24 h. Thereafter the positive cells decreased markedly from 24 h after PDT.
method and destroyed tumor cells were observed sporadically (Fig. 7). In immunocytochemical detection with MoAb M30, M30-positive cells increased from 2 h after PDT, increased rapidly from 8 h and peaked at 24 h. Therefore, the results of the investigation with MoAb M30 suggested that the induction of apoptosis of HSG by PDT occurred gradually from 2 h after laser irradiation, becoming marked from 8 to 24 h after irradiation. On the other hand, the in vivo examination showed M30-positive cells increased markedly from 0.5 h after laser irradiation and tended to further increase at 1 h and the number of positive cells peaked at 2 h. Therefore, a marked induction of apoptosis occurred from 30 min to 2 h after irradiation. The results of immunohistochemical examination in vivo suggested that the induction of apoptosis was obtained from the period earlier than that observed in HSG cells by PDT in vitro, but a marked decrease in M30-positive cells was observed in vitro and in vivo from 24 h after laser irradiation. Since TUNEL-positive cells showing DNA fragmentation by the in vivo TUNEL method increased from 24 h after irradiation, these findings suggested that MoAb M30 could detect the induction of apoptosis earlier than the TUNEL method. It is generally known that MoAb M30 reacts specifically with decomposition products of CK 18 by activated caspase.3;4 Both CK 18 and CK 8 are important constitutive components of the intermediate filament in the glandular cells and glan-
Apoptosis of HSG cells by PDT
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Figure 5 Histological apoptosis of HSG cells induced by PDT. M30-positive cells increased markedly in Group 1 from 0.5 h after PDT and the number of the positive cells tended to further increase in Group 2 and peaked in Group 3 from 2 h after PDT. The positive cells were observed continuously in Group 5 until 24 h after PDT. Thereafter M30-positive cells themselves decreased.
Figure 4 Induction of apoptosis of HSG cells by PDT in vitro. HSG cells were treated 4 h (a), 8 h (b) and 24 h (c) after PDT. After fixation with a mixture of 95% ethanol and 5% acetic acid, the cells were stained by immunocytochemical staining with MoAb M30 (original magnification 50·).
dular tumor cells,8 and their presence indicated the onset of apoptosis in HSG cells used in the present study.5 Since the decomposition product of CK 18 is produced by activated caspase and MoAb M30 reacts with the decomposition product when apoptosis was induced in cells with CK 18 in the skeletal system, the early detection of apoptosis is possible.4–7 In PDT the pathological site is damaged by the active oxygen produced by exciting the tetrapyrrol ring of the photosensitizer by laser light. In per-
Figure 6 Induction of apoptosis of HSG cells by PDT in vivo. Implanted tumors of HSG cells were treated 2 h (a), and 24 h (b) after PDT. The tissues were fixed in 10% neutralized buffered formalin and the cells were stained by immunohistochemical staining with MoAb M30 (original magnification 50·).
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T. Kaneko et al. tizer, is effective in inducing apoptosis in tumor cells.
Acknowledgements
Figure 7 Induction of apoptosis of HSG cells by PDT and TUNEL-positive cells increased in Group 5 from 24 h after PDT and the destroyed tumor cells, were observed sporadically (original magnification 50·).
forming PDT effectively, it is recommended that the absorption zone of photosensitizers should be at wavelengths longer than 600 nm to avoid the absorption zone of living substances such as hemoglobin in the blood. Photosensitizers must also have excellent tumor affinity, distribute uniformly within the tumors and be excreted rapidly from normal tissues. The distribution of the early onset of apoptosis in human salivary gland tumor, which was examined in the present study, was observed in all tumor cells, including deep areas of the tumor. Furthermore the onset of apoptosis at the central part of tumor was delayed with time compared with the superficial areas of the tumor, No increased induction of apoptosis was seen in normal tissues. Therefore NPe6, a photosensitizer with a long absorption wavelength (664 nm), used in the present study is considered to distribute uniformly to the tumor tissues and to penetrate the deep parts of tumor without absorption in normal tissues and to be quickly excreted from the normal tissues. In conventional PDT with porphyrin derivatives,1;7 tumor is irradiated by a laser beam 48–72 h after their administration, but in PDT with NPe6 used in the present study, the tumor is irradiated by a diode laser beam only 2 h after administration and the marked cytocidal effect of NPe6 on tumor was confirmed. PDT-inducing apoptosis in all tumor cells and totally destroying tumors would be ideal. The results of the present study suggest the possibility that PDT with NPe6, a new photosensi-
The authors are indebted to Professor J. Patrick Barron of the International Medical Communications Center of Tokyo Medical University for his review of this manuscript. The authors also thank Professor H. Kato (Department of Surgery, Tokyo Medical University) and Professor T. Okunaka (International University of Health and Welfare) for their excellent advice. This research was supported in part by a Grant from the Intractable Disease Research Center of Tokyo Medical University.
References 1. Noodt BB, Berg K, Stokke T, Peng Q, Nesland JM. Different apoptotic pathways are induced from various intracellular sites by tetraphenylporphyrins and light. Br J Cancer 1999;79:72–81. 2. Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis. Biochim Biophys Acta 1998;1366:151–65. 3. Caulin C, Salvesen GS, Oshima RG. Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 1997;138: 1379–94. €lgen W, Bjo €rklund V, et al. Immunocytochem4. Leers MPG, Ko ical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 1999;187:567–72. 5. Kusama K, Jiang Y, Toguchi M, et al. Use of the monoclonal antibody M30 for detecting HSG cell apoptosis. Anticancer Res 2000;20:151–4. 6. Kusama K, Jiang Y, Kaneko T, et al. Immunohistochemical detection of cytokeratin 18 and its neo-epitope in human salivary glands and pleomorphic adenomas. Anticancer Res 2000;20:2485–8. 7. Kaneko T, Chiba H, Yasuda T, Kusama K. Detection of transplanted human salivary gland tumor cell apoptosis induced by photodynamic therapy. Hosp Dent 2003; 15:41–3. 8. Berkovitz BKB, Barrett AW. Cytokeratin intermediate filaments in oral and odontogenic epithelia. Bull Group Int Rech Sci Stom Odontol 1998;40:4–23. 9. Aizawa K, Okunaka T, Ohtani T, et al. Localization of monoL-aspartyl chlorin e6 (NPe6) in mouse tissues. Photochem Photobiol 1987;46:789–93. 10. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of Programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493– 501. 11. Institute of Laboratory Animal Resources. Guide for the care and use of laboratory animals. Washington: National Academy Press; 1996.
http://intl.elsevierhealth.com/journals/oron/
Detection of photodynamic therapy-induced early apoptosis in human salivary gland tumor cells in vitro and in a mouse tumor model Tadayoshi Kanekoa,*, Hiroshige Chibaa, Takashi Yasudaa, Kaoru Kusamab a
Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan b Deparment of Oral Pathology, Meikai University School of Dentistry, Sakado, Saitama, Japan Received 11 December 2003; accepted 31 January 2004
Available online KEYWORDS
Summary We studied the detection of apoptosis of malignant human salivary gland tumor cells induced by photodynamic therapy (PDT) using the photosensitizer monoL-aspartyl chlorin e6 (NPe6) in vitro and in vivo in mice receiving transplanted human salivary gland tumor (HSG) cells. An immunohistocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin (CK) 18 by activated caspase, was applied to detect the apoptosis of HSG cells induced by PDT. Significant amounts of immunoreactive products were observed in the cytoplasm of HSG cells after PDT. In vitro, M30positive cells increased from 2 h after PDT, increased rapidly from 8 h and reached a peak 24 h after PDT. In vivo, a peak of early apoptosis was confirmed two hours after PDT. In comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, the destroyed tumor cells were observed sporadically 24 h after PDT. These results suggest that immunohistocytochemical staining with the MoAb M30 may be useful for detecting early apoptosis induced by PDT. Futhermore, PDT using NPe6 is effective in inducing apoptosis of HSG cells at an early stage, which suggests the possibility of the therapy being ideal for treatment of human malignant neoplasms. c 2004 Published by Elsevier Ltd.
Photodynamic therapy; Human salivary gland tumor cells; M30
Introduction Photodynamic therapy (PDT) may result in either apoptotic or necrotic cell death.1 PDT may interact * Corresponding author. Tel.: +81-33342-6111, fax: +8133342-1723. E-mail address: [email protected] (T. Kaneko).
1368-8375/$ - see front matter c 2004 Published by Elsevier Ltd. doi:10.1016/j.oraloncology.2004.01.007
with many pathways but it is unclear exactly what is responsible for early apoptosis following PDT. Many stimuli may trigger apoptosis. These stimuli interact with mitochondria, which are thought to be key regulators of apoptosis by activations of the caspase cascade.2 The monoclonal antibody M30 reacts specifically with the product resulting from the cleavage of CK 18 by activated caspase3;4 and is
788 known to be useful for detection of early apoptosis in human salivary glands and tumors derived from salivary glands.4–8 We examined the early apoptosis-inducing activities of PDT in combination with NPe6,9 a relatively new photosensitizer, for HSG cells by the immunohistocytological method using MoAb M30 and compared the results with those obtained using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method.10
T. Kaneko et al.
In vitro study of photosensitizer properties Cell culture A human salivary gland tumor HSG cell line was maintained in monolayer cultures at 37 C in RPMI 1640 medium supplemented with 10% heatinactivated FBS in a humidified 5% CO2 atmosphere. Assay for cytotoxic and antitumor activities
Materials and methods Materials The following chemicals and reagents were obtained from the indicated companies: NPe6 (Meiji Seika, Ltd., Tokyo, Japan, structure shown in Fig. 1); diode laser (Matsushita, Ltd., Tokyo, Japan); RPMI 1640 medium (GIBCO, Grand Island, NY, USA); fetal bovine serum (FBS, GIBCO-BRL , Life Technologies Inc., Gaithersburg, MD, USA); WST-1, 1Methoxy PMS (WST, Dojin Chem. Ind., Kumamoto, Japan); MoAb M30 (Boehringer Mannheim, GmbH, Mannheim, Germany); MoAb DC-10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); streptavidin-peroxidase (GIBCO-BRL , Life Technologies Inc.); biotinylated horse anti-mouse Ig G (H + L) antibody (Vector Laboratories lnc., Burlingame, CA, USA); 3,30 -diaminebenzidine tetrahydrochloride (DAB, Sigma Chemical Industries., St. Louis, MO, USA); a TACS in situ apoptosis detection kit (Trevigen, Inc., Gaithersburg, MD, USA).
The relative viable cell number was determined by the WST method. Near-confluent HSG cells grown in 96-microwell plates (Falcon, flat bottom, treated polystyrene, Becton Dickinson and Company., Franklin Lakes, NJ, USA) were incubated for 24 h, and then were incubated for 2 more hours with NPe6 at 0, 1.25, 2.5, 5.0, 10, 20 and 40 lg/ml in RPMI 1640 medium supplemented with 10% FBS, washed once with phosphate-buffered saline (PBS, 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.4). PDT was performed with a diode laser (absorption peak: 664 nm, output: 100 mW/cm2 and total radiation: 10 J/cm2 ) and the materials were then incubated in the dark for 24 more hours after PDT in RPMI 1640 medium supplemented with 10% FBS, and then incubated for 4 h with 0.2 mg/ml WST. After the medium was removed, the cells were lysed with 100 ll DMSO and the relative viable cell number was determined by measuring the absorbance at 650 nm of the cell lysate using a model 550 microplate reader (Bio-Rad laboratories, Inc., Hercules, CA, USA) Detecting apoptosis induced by PDT
Figure 1 NPe6 was used as a photosensitizer. NPe6 has a ring of 4 pyrroles and asparatic acid combining with the D ring. The molecular weight is 799.7 and the absorption peak is 664 nm. NPe6 is a compound with a high affinity for tumor.
A chamber slide (Nalge Nunc International, Lab-Tek Brand Products., Naperville, IL, USA) with 5 · 104 /ml of HSG cells was incubated, then 2 h later 10 lg/ml of NPe6 was added, and PDT was performed under the same conditions as described above. The samples were allocated to experimental Group 1 (0.5 h after laser irradiation), Group 2 (at 1 h), Group 3 (at 2 h), Group 4 (at 4 h), Group 5 (at 8 h), Group 6 (at 24 h), Group 7 (at 48 h) and control group (no laser irradiation). Cultured cells on chamber slides were fixed at those times with a mixture of 95% ethanol and 5% acetic acid for 10 min at 4 C. Thereafter the samples were stained immunocytochemically. The mean numbers of M30-positive cells in five different fields under a microscope (5 · 3.3 times) in each group were calculated and were compared.
Apoptosis of HSG cells by PDT
Animal experiments Animals Male BALB/c-nude mice aged 4 weeks were used as experimental animals. All animals received good care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”11 and all experiments were carried out in accordance with protocols approved by the local Experimental Animal Research Committee.
789 (1:200) at room temperature for 30 min. After the samples were washed with PBS three times, the samples were color-developed with DAB solution and washed with PBS. After washing with water, the nuclei were stained with Mayer hematoxylin, washed with water, dehydrated and mounted. For the activation of antigenicity, the samples were treated beforehand with 0.01M-citrate buffer (pH 6.0) by microwave for 15 min.
Detection of apoptotic cells by DNA nick end labeling of tissue sections
Detecting apoptosis induced by PDT in vivo Cell suspensions of human salivary gland tumor cells (5 · 106 ) were injected aseptically into the subcutaneous tissues of the femur of male BALB/cnude mice (mean body weight: 25 g) aged 4 weeks. NPe6 (10 mg/kg) was injected intravenously into the tissues 2 weeks after transplantation of tumor cells, by which time the tumors had reached about 5 mm in diameter, and 2 h later tumor was irradiated with a diode laser (664 nm, 100 J/cm2 ). The mice were allocated to Group 1 (sacrificed at 0.5 h after laser irradiation), Group 2 (at 1 h), Group 3 (at 2 h), Group 4 (at 4 h), Group 5 (at 24 h), Group 6 (at 72 h) and control group (sacrificed without laser irradiation) and samples were collected from each group. These test samples were fixed in 10% buffered neutral formalin, embedded in paraffin and thin-sectioned in the routine manner. After deparaffinization, the thin-sections were reacted with methanol containing 0.3% H2 O2 at room temperature for 15 min to inhibit endogenous peroxidase. Thereafter the samples were stained immunohistochemically. The mean numbers of M30-positive cells in five different fields under a microscope (5 · 3.3 times) in each group were calculated and were compared. DNA fragmentation was also investigated in the in vivo group by the TUNEL method.
In order to demonstrate the apoptosis of cells induced by PDT, DNA fragmentation was visualized by the modified method using a TACS in situ apoptosis detection kit. Cells treated by PDT on slides were fixed with 10% neutralized buffered formalin for 10 min at room temperature and after washing with water, each slide was dried for 30 min at 56 C. The slides were rinsed with PBS and treated with 10 lg/ml proteinase K for 5 min at room temperature. To block endogenous peroxidase activity, the slides were immersed in 2% H2 O2 solution for 5 min at room temperature. After washing in PBS and pretreatment with labeling buffer, each slide was incubated with labeling mixture (TdT, biotin-dUTP) for 60 min at 37 C. After stopping the reaction, each slide was treated with 2% bovine serum albumin in PBS (BSA-PBS) for 15 min to block nonspecific reactions, and then incubated with diluted streptavidin-peroxidase (1:200). The slides were washed with PBS three times and immersed for 10 min in 0.05% 3,30 -diaminebenzidine tetrahydrochloride (DAB) in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% H2 O2 . Each slide was counterstained with Mayer’s hematoxylin and mounted.
Results and discussion Immunologic procedure After samples were washed with phosphate buffer solution (PBS), the samples were reacted with 2% bovine serum albumin BSA (bovine serum albumin)-PBS at room temperature for 15 min to block non-specific reaction and reacted with MoAb M30 (1:30) for 60 min. After the samples were washed with PBS three times, they were reacted with anti-mouse IgG (H + L) horse antibody (1:200) at room temperature for 30 min. After the samples were washed with PBS three times, the samples were reacted with streptavidin-peroxidase solution
We have previously shown that the distribution of CK 18 in the salivary glands and pleomorphic adenomas and extent of apoptosis in them were determined using a MoAb M30, which reacts with the caspase cleavage neo-epitope in CK 18 exposed during early apoptosis.6 We developed this to detect the apoptosis of HSG cells induced by PDT.7 To establish whether MoAb M30 could detect activity of the early apoptosis induced by PDT, we prepared an in vivo mouse model of transplanted HSG cells and irradiated the HSG cells both in vivo and in vitro.
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Figure 2 Number of viable HSG cells determined in the laser-irradiation groups () compared with the non-laserirradiation group () by the WST method. Each value represents a mean ± SD for triplicate assays.
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The antitumor activity by PDT against HSG cells was clearly obtained concentration-dependently in the range of 5–20 lg/ml of NPe6 in the irradiation groups as compared with the non-irradiation group. There were only 8.1% viable cells following PDT after treatment with 20 lg/ml of NPe6, thus marked antitumor activity was obtained (Fig. 2). Concerning detection of apoptosis induced by PDT in vitro, M30- positive cells increased in Group 3 from 2 h after PDT, increased rapidly in Group 5 from 8 h and reached a peak in Group 6 at 24 h. Thereafter the positive cells decreased markedly and were nearly the same in Group 7 at 48 h after PDT as those in Group 4 at 4 h. M30-positive cells decreased from 24 h after PDT (Figs. 3 and 4). In vivo, M30-positive cells increased markedly in Group 1 from 0.5 h after PDT and were distributed more entirely throughout tumor tissues than in the control group. The number of positive cells tended to further increase in Group 2 and peaked in Group 3. Positive cells were observed continuously in Group 5 until 24 h after PDT. Apoptosis was observed in the whole tumor area including the deep part of the tumor by 24 h after PDT. Thereafter M30-positive cells themselves decreased (Figs. 5 and 6). A tendency to delayed onset of apoptosis was observed in the deep part of the tumor compared to the superficial parts of the tumor. In the induction of apoptosis in vivo, TUNELpositive cells increased in the implanted tumor part in Group 5 from 24 h after PDT by the TUNEL
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Figure 3 Cytological apoptosis of HSG cells induced by PDT. M30-positive cells increased in Group 3 from 2 h after PDT, increased rapidly in Group 5 from 8 h and reached a peak in Group 6 at 24 h. Thereafter the positive cells decreased markedly from 24 h after PDT.
method and destroyed tumor cells were observed sporadically (Fig. 7). In immunocytochemical detection with MoAb M30, M30-positive cells increased from 2 h after PDT, increased rapidly from 8 h and peaked at 24 h. Therefore, the results of the investigation with MoAb M30 suggested that the induction of apoptosis of HSG by PDT occurred gradually from 2 h after laser irradiation, becoming marked from 8 to 24 h after irradiation. On the other hand, the in vivo examination showed M30-positive cells increased markedly from 0.5 h after laser irradiation and tended to further increase at 1 h and the number of positive cells peaked at 2 h. Therefore, a marked induction of apoptosis occurred from 30 min to 2 h after irradiation. The results of immunohistochemical examination in vivo suggested that the induction of apoptosis was obtained from the period earlier than that observed in HSG cells by PDT in vitro, but a marked decrease in M30-positive cells was observed in vitro and in vivo from 24 h after laser irradiation. Since TUNEL-positive cells showing DNA fragmentation by the in vivo TUNEL method increased from 24 h after irradiation, these findings suggested that MoAb M30 could detect the induction of apoptosis earlier than the TUNEL method. It is generally known that MoAb M30 reacts specifically with decomposition products of CK 18 by activated caspase.3;4 Both CK 18 and CK 8 are important constitutive components of the intermediate filament in the glandular cells and glan-
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Figure 5 Histological apoptosis of HSG cells induced by PDT. M30-positive cells increased markedly in Group 1 from 0.5 h after PDT and the number of the positive cells tended to further increase in Group 2 and peaked in Group 3 from 2 h after PDT. The positive cells were observed continuously in Group 5 until 24 h after PDT. Thereafter M30-positive cells themselves decreased.
Figure 4 Induction of apoptosis of HSG cells by PDT in vitro. HSG cells were treated 4 h (a), 8 h (b) and 24 h (c) after PDT. After fixation with a mixture of 95% ethanol and 5% acetic acid, the cells were stained by immunocytochemical staining with MoAb M30 (original magnification 50·).
dular tumor cells,8 and their presence indicated the onset of apoptosis in HSG cells used in the present study.5 Since the decomposition product of CK 18 is produced by activated caspase and MoAb M30 reacts with the decomposition product when apoptosis was induced in cells with CK 18 in the skeletal system, the early detection of apoptosis is possible.4–7 In PDT the pathological site is damaged by the active oxygen produced by exciting the tetrapyrrol ring of the photosensitizer by laser light. In per-
Figure 6 Induction of apoptosis of HSG cells by PDT in vivo. Implanted tumors of HSG cells were treated 2 h (a), and 24 h (b) after PDT. The tissues were fixed in 10% neutralized buffered formalin and the cells were stained by immunohistochemical staining with MoAb M30 (original magnification 50·).
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T. Kaneko et al. tizer, is effective in inducing apoptosis in tumor cells.
Acknowledgements
Figure 7 Induction of apoptosis of HSG cells by PDT and TUNEL-positive cells increased in Group 5 from 24 h after PDT and the destroyed tumor cells, were observed sporadically (original magnification 50·).
forming PDT effectively, it is recommended that the absorption zone of photosensitizers should be at wavelengths longer than 600 nm to avoid the absorption zone of living substances such as hemoglobin in the blood. Photosensitizers must also have excellent tumor affinity, distribute uniformly within the tumors and be excreted rapidly from normal tissues. The distribution of the early onset of apoptosis in human salivary gland tumor, which was examined in the present study, was observed in all tumor cells, including deep areas of the tumor. Furthermore the onset of apoptosis at the central part of tumor was delayed with time compared with the superficial areas of the tumor, No increased induction of apoptosis was seen in normal tissues. Therefore NPe6, a photosensitizer with a long absorption wavelength (664 nm), used in the present study is considered to distribute uniformly to the tumor tissues and to penetrate the deep parts of tumor without absorption in normal tissues and to be quickly excreted from the normal tissues. In conventional PDT with porphyrin derivatives,1;7 tumor is irradiated by a laser beam 48–72 h after their administration, but in PDT with NPe6 used in the present study, the tumor is irradiated by a diode laser beam only 2 h after administration and the marked cytocidal effect of NPe6 on tumor was confirmed. PDT-inducing apoptosis in all tumor cells and totally destroying tumors would be ideal. The results of the present study suggest the possibility that PDT with NPe6, a new photosensi-
The authors are indebted to Professor J. Patrick Barron of the International Medical Communications Center of Tokyo Medical University for his review of this manuscript. The authors also thank Professor H. Kato (Department of Surgery, Tokyo Medical University) and Professor T. Okunaka (International University of Health and Welfare) for their excellent advice. This research was supported in part by a Grant from the Intractable Disease Research Center of Tokyo Medical University.
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