Photodiagnosis and Photodynamic Therapy (2006) 3, 247—258

Apoptosis and expression of cytokines triggered by pyropheophorbide-a methyl ester-mediated photodynamic therapy in nasopharyngeal carcinoma cells K.M. Li a,1, X. Sun a,2, H.K. Koon a, W.N. Leung b,3, M.C. Fung c, R.N.S. Wong a, Maria L. Lung d, C.K. Chang e, N.K. Mak PhD a,∗ a

Department of Biology, Hong Kong Baptist University, 224 Waterloo Road, Hong Kong, China School of Chinese Medicines, Hong Kong Baptist University, China c Department of Biology, Chinese University of Hong Kong, China d Department of Biology, Hong Kong University of Science and Technology, Hong Kong, China e Department of Chemistry, Hong Kong University of Science and Technology, Hong Kong, China b

Available online 17 October 2006 KEYWORDS Apoptosis; Nasopharyngeal carcinoma; Photodynamic therapy; Mitochondrial membrane potential; Pyropheophorbide-a methyl ester

Summary The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5—2 ␮M, MPPa-mediated a drug dosedependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1␤, IL-6, and TNF-␣) and beta-chemokines (MIP-1␤, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma. © 2006 Elsevier B.V. All rights reserved.

Abbreviations: ␺m, mitochondrial membrane potential; CNX, calnexin; ER, endoplasmic reticulum; MPPa, pyropheophorbide-a methyl ester; NPC, nasopharyngeal carcinoma; PDT, photodynamic therapy; ROS, reactive oxygen species ∗ Corresponding author. Fax: +852 3411 5995. E-mail address: [email protected] (N.K. Mak). 1 Present address: Department of Biochemistry, University of Hong Kong, China. 2 Present address: The Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, 243 Charles Street, Boston, Massachusetts 02114, USA. 3 Present address: The Chinese University of Hong Kong—–Tung Wah Group of Hospitals Community College, China. 1572-1000/$ — see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2006.09.001

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Introduction Nasopharyngeal carcinoma (NPC) is a tumor arising from the epithelium of the nasopharynx. NPC is endemic in southern China and southeast Asia. Radiotherapy is the primary treatment for NPC, and chemotherapy is usually used for advanced NPC. In the past two decades, photodynamic therapy (PDT) was developed as an effective modality for the treatment of various malignant diseases [1]. During PDT, the photosensitizer is preferentially absorbed and retained in the malignant tissues. The sensitizer is then photoactivated by light of an appropriate wavelength. Subsequent photoactivation of the photosensitizer results in tumour cell death. In view of the differences of tumour properties among the various tumour cell types, finding a suitable photosensitizer is crucial in improving the efficacy of PDT. Most of the newer photosensitizers are cyclic tetrapyrroles comprising substituted derivatives of porphyrin, chlorine, and bacteriochlorin [2,3]. Photosensitizers with other types of chemical structure, such as phthalocyanines, hypericin, rhodamine, methylene blue, and derivatives of these compounds, have also been studied [3—7]. Of these various photosensitizers, photosensitizers prepared by partial synthesis starting from the abundant natural precursors, protoheme and the chlorophylls have received much attention [2]. In plants, chlorophylls play a role in harvesting light and transferring the light energy into chemical energy. The unique structure and properties of chlorophylls also make them and their derivatives good candidates as photodynamic sensitizers. Talaporfin sodium, a recently developed photosensitizer prepared from plant chlorophyll, was tested clinically for the treatment of patients with a broad range of treatment-resistant malignancies [8,9]. The skin photosensitivity caused by talaporfin was found to disappear faster than for some of the existing photosensitizers in the market. Pyropheophorbide-a methylester (MPPa), also known as PPME [10], is a semi-synthetic photosensitizer derived from chlorophyll a. MPPa PDT triggers apoptosis in colon, nasopharyngeal and lung carcinoma cells [10—12]. The effect of MPPa PDT on other types of tumour has not been studied. In addition to the direct cytotoxic effect on tumour, PDT also induces a complex immune response that may potentiate anti-tumour immunity. Tumour-derived cytokines have also been implicated to play a role in PDT-induced anti-tumour immunity by regulating host immune response involving both lymphoid and non-lymphoid cells. In this study, we herein examine the mechanisms of induction of apoptosis and the expression of cytokines genes in MPPa

K.M. Li et al. PDT-treated NPC cells. We demonstrate that MPPa PDT induces apoptosis of NPC via mitochondriamediated apoptotsis pathway.

Materials and methods Photosensitizer MPPa was purchased from Sigma—Aldrich Co (USA). MPPa has a molecular structure of C34 H36 N4 O3 . The purity of MPPa used in this study was 95%. Its absorption and fluorescence spectra have been described elsewhere [11]. A stock solution of 1 mM was prepared in DMF and the stock solution was stored in the dark at 4 ◦ C.

Cell culture The HONE-1 cell line, derived from a patient with nasopharyngeal carcinoma [13], was maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Gibco) and antibiotics (50 ␮g/ml penicillin G, 50 ␮g/ml streptomycin, Gibco). Cells were incubated at 37 ◦ C in a humidified atmosphere in a 5% CO2 incubator.

Localization of MPPa in HONE-1 cells Subcellular photosensitizer localization was determined using laser scanning confocal microscopy [14]. Briefly, overnight cultured HONE-1 cells (1 × 105 cells in 35 mm petri dishes) were treated with MPPa (2 ␮M) for 24 h at 37 ◦ C. The cells were then washed with fresh medium and stained with the specific organelle probes. Before microscopic examination, the cells were stained with probes specific for mitochondria (100 nM, Mitotracker M7514, Molecular Probes, USA) for 20 min, Golgi apparatus (2 ␮M, D-3521, Molecular Probes) for 20 min, or lysosomes (200 nM, Lysotracker L-7526, Molecular Probes) for 30 min. Confocal images of the cells were visualized using a laser scanning confocal microscope (Zeiss, LSM 510, Germany). The optimal pinhole size of 120 ␮m was selected to exclude fluorescence light emitted from out-offocus planes above and below the focusing plane. An objective with a magnification of 63× was used for image capture. An argon/krypton laser line with a wavelength of 488 nm was used for excitation of the organelle probes and the HeNe laser line was used for the excitation of MPPa. The band-pass filter (BP505-550 nm) and long-pass filter (LP 650 nm) were used at the emission end for the detection of

Apoptosis and expression of cytokines triggered by MPPa the organelle probes and MPPa, respectively. Fluorescence images were analyzed using the software LSM510 (Zeiss).

Cytotoxicity assay The photocytotoxicity of MPPa was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyltetrazolium bromide] reduction assay [14]. Briefly, HONE-1 cells (1 × 104 cells/well) in 96-well flat bottomed tissue culture plates were incubated with various concentrations of MPPa (0.5—2 ␮M), three replicates per treatment, for 24 h at 37 ◦ C. The cells were washed with PBS and exposed to light (0—5 J/cm2 ) emitted from a 400 W tungsten lamp equipped with a heat isolation filter and a narrow band light filter suitable for MPPa (667 ± 5 nm). PDTtreated NPC cells were then incubated at 37 ◦ C in a 5% CO2 incubator for 24 h. Viability was then determined by incubating the cells with MTT (Sigma) solution. The optical density (OD) of DMSO dissolved formazan crystals was measured at wavelengths of 540 nm and 690 nm by an iEMS Analyzer (Labsystems, Type 1401, Finland). The percentage of survival was calculated by using the following equations: Survival(%) = where

ODTreatment group × 100 ODControl group

OD = OD540nm − OD690nm .

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30 min before cell harvesting. The washed cells were then resuspended in PBS containing 15 nM TMRE. The change of mitochondrial membrane potential was analyzed at 15 min post PDT using a FACScan Flow Cytometer (Becton Dickinson, USA) with excitation setting at 488 nm. The fluorescence signals were analyzed using the Cell Quest software.

Detection of cytochrome c Flow cytometric method was used to detect the release of cytochrome c in NPC cells after PDT [14]. Briefly, MPPa PDT-treated NPC cells were trypsinized, fixed with paraformaldehyde (3% in PBS for 15 min) and then stained with anti-cytochrome c (PharMingen, USA; 1:500 in PBS supplemented with 2% normal goat serum and 0.1% Triton-X,) and FITCconjugated secondary antibody (PharMingen). Fluorescence intensity of the stained cells was then measured using a FACScan Flow Cytometer (Becton Dickinson) with excitation at 488 nm. About 1 × 104 cells were analyzed in triplicate runs and the data were analyzed by using Cell Quest Software.

Measurement of caspase activity The activities of caspase-3 and caspase-9 in MPPa PDT-treated HONE-1 cells were determined using BD ApoAlert caspase fluorescent assay kits (Becton Dickson). All procedures were performed according to the instructions provided by the manufacturer.

Nuclear staining of apoptotic bodies HONE-1 cells (1 × 105 cells) in 35 mm petri dishes were incubated with MPPa (2 ␮M) for 24 h at 37 ◦ C. The cells were then washed and exposed to light at light doses of 0.4—1 J/cm2 . MPPa PDT-treated HONE-1 cells were then incubated at 37 ◦ C for 16 h. The cells were stained with Hoechst 33258 (0.1 ␮g/ml) dye (Fluka, Germany) for 15 min. Fluorescence images of the stained cells were observed and captured using an Imaging Analyzer Fluorescence Microscope (Zeiss, Germany).

Flow cytometric analysis of mitochondrial membrane potential Flow cytometric method was used to monitor the change of mitochondria membrane potential (m) of PDT-treated HONE-1 cells [14]. Briefly, HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then washed and irradiated at the light dose of 0.4—1 J/cm2 . The voltagesensitive dye tetramethyl rhodamine ethyl ester (TMRE, 150 nM, Molecular Probes, USA) was added

Western blotting analysis Western blot was used to analyze the expression of ER resident stress proteins calnexin (CNX), Grp78 (Bip), and Grp94. Briefly, MPPa PDT-treated HONE-1 cells were lysed with lysis buffer (250 mM Tris—HCl, pH 8, 1% NP-40, and 150 mM NaCl). The lysate was then centrifuged at 14,000 rpm for 30 min at 4 ◦ C to remove cell debris. Protein concentration of the cell lysate was measured using a protein assay kit (Bio-Rad, USA). Fifty micrograms of total cellular protein in the cell lysate was subjected to 10% SDS-PAGE electrophoresis using the PROTEAN® II electrophoresis system (Bio-Rad). The separated proteins were then electrophoretically blotted onto Immuno-Blot PVDF membraneTM (Bio-Rad). The primary antibodies were anti-calnexin (SPA-860, Stressgen) and antiKDEL (SPA-827, Stressgen, Canada). The anti-KDEL monoclonal was raised against the synthetic peptide SEKDEL, based on the rat Grp78 (amino acids 649—654). This antibody identifies Grp78 (Bip) and Grp94. Protein bands were visualized on a Biomax X-

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Table 1

Summary of PCR primer sequences, PCR conditions and product sizes

Genes

Primer sequence

Annealing temperature (◦ C)

MgCl2 (mM)

Amplification cycles

Amplified fragments (base pairs)

IL-1␤

5 -CTT CAT CTT TGA AGA AGA ACC TAT CTT CTT-3 5 -AAT TTT TGG GAT CTA CAC TCT CCA GCT GT-3

60

1.5

33

331

IL-6

5 -ATG AAC TCC TTC TCC ACA AGC GC-3 5 -GAA GAG CCC TCA GGC TGG ACT G

60

1.5

26

621

TNF-␣

5 -CGG GAC GTG GAG CTG GCC GAG GAG-3 5 -CAC CAG CTG GTT ATC TCT CAG CTC-3

66

1.5

30

355

MIP-1␤

5 -TGT CTC TCC TCA TGC TAG TA-3 5 -GTA CTC CTG GAC CCA GGA T-3

60

1.5

30

233

MPIF-1

5 -CTC CGT GGC TGC CCT CTC C-3 5 -TGG TGG CTG GCA ACT TGT GTC-3

60

1.5

33

393

MPIF-2

5 -GGC CTG ATG ACC ATA GTA ACC A-3 5 -GGT TTG GTT GCC AGG ATA TCT C-3

60

1.5

33

345

RANTES

5 -ATG AAG GTC TCC GCG GCA CGC CT-3 5 -CTA GCT CAT CTC CAA AGA GTT G-3

60

1.5

30

276

GAPDH

5 -ACC ACA GTC CAT GCC ATC AC-3 5 -TCC ACC ACC CTG TTG CTG TA-3

56

1.5

20

452

ray film (Kodak, Japan) using the enhanced chemiluminescence ECLTM Western Blotting Detection Reagents (Amersham Biosciences, RPN2209, UK).

Reverse transcription polymerase chain reaction (RT-PCR) MPPa PDT-induced cytokine gene expression was detected using RT-PCR [15]. Briefly, total cellular RNA was prepared from MPPa PDT-treated HONE-1 cells using the guanidinium isothiocyanate-cesium chloride ultracentrifugation method. RNA samples with the OD260 :OD280 ratio between 1.9 and 2.1 were used for the analysis of cytokine gene expression. For synthesis of first-strand cDNAs, 3 ␮g of total RNA in 30 ␮l diethyl pyrocarbonate (DEPC, Sigma)-treated double distilled water was incubated at 65 ◦ C for 5 min and then chilled on ice immediately. The heat denatured total RNA was used to perform the reverse transcription reaction in a 60 ␮l reaction, containing 200 U of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Gibco), 0.2 mM of each dNTP, 0.1 ␮g Oligo (dT)12—18 (Pharmacia, Sweden), 1 × firststrand buffer (Gibco), 10 mM dithiothreitol, and 40 U RNaseOUT (Invitrogen, USA). The reaction mixture with final volume of 60 ␮l was incubated at 37 ◦ C for 1 h. The reaction mixture was then diluted 10-fold with ddH2 O and boiled for 5 min to denature the first strand DNA from the RNA template and then chilled on ice immediately. The diluted sam-

ples were stored at −70 ◦ C until use. For PCR, 30 ␮l of master mix containing 1 × reaction buffer IV, 0.2 mM of each dNTP, 1.25 units ThermoprimePLUS DNA polymerase (ABgene, UK), and 50 pmol of primers (Table 1) in the defined MgCl2 concentration were added into 20 ␮l of boiled RT sample containing 0.1 ␮g of total RNA. PCR was performed by initial denaturation at 94 ◦ C for 30 s, annealing at defined temperature for 30 s, extension at 72 ◦ C for 1 min. Ten microliters of PCR-amplified products were electrophoresed on a 2% agarose gel with 0.25 ␮g/ml ethidium bromide. After gel electrophoresis, the gel was visualized under UV illumination with an ultraviolet transilluminator. GAPDH was used to normalize RNA sample quantitation and all of the analyses were performed for two to three times.

Results Photocytotoxicity of MPPa Photocytotoxicity of MPPa on HONE-1 cells was determined by using the MTT reduction assay. HONE-1 cells were treated with different MPPa concentrations (0—2 ␮M) and different light doses (0—5 J/cm2 ). As shown in Fig. 1, MPPa killed HONE1 cells in a drug- and light-dose dependent manner. When 2 ␮M of MPPa and 1 J/cm2 of light were used in combination, over 95% of HONE-1 cells were

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Localization of MPPa

Figure 1 Photocytotoxicity of MPPa on HONE-1 cells. HONE-1 cells were treated with various concentrations of MPPa for 24 h and then irradiated with various light doses. MTT cytotoxicity assay was performed 24 h after PDT. Results represent mean ± S.D. of three replicates.

killed. In the absence of light irradiation, MPPa was not cytotoxic to the HONE-1 cells. To investigate the mode of cell death after MPPa PDT treatment, MPPa PDT-treated HONE-1 cells were then stained with the nuclear dye Hoechst 33258. Apoptotic bodies, as judged by the appearance of condensed and lobulated nuclei, were clearly seen in HONE-1 cells after MPPa PDT treatment (Fig. 2).

To determine the subcellular localization of MPPa, HONE-1 cells were co-stained with MPPa and three organelle-specific probes. Fig. 3a shows the confocal images of stained HONE-1 cells. MPPa was found mainly in the cytoplasm (red channel). Nuclear localization of MPPa was not seen in the stained HONE-1 cells. Yellow fluorescence indicated the overlap of MPPa and the organelle probes (green channel). The results suggest that MPPa mainly localizes in the mitochondria and Golgi body; little MPPa localizes in lysosomes. This observation was further confirmed by analyzing the fluorescence intensity profile drawn across the cell. Fig. 4 shows the representative profiles of the fluorescence intensity of MPPa and the corresponding organelle probes (Mitotracker, Golgi body probe, and Lysotracker). The fluorescence profile of MPPa overlaps well with that of Mitotracker and Gogli body probe. The results indicate that MPPa is localized mainly in the mitochondria and Golgi body, and to a lesser extent in lysosomes.

Loss of mitochondrial membrane potential and release of the cytochrome c after PDT Collapse of mitochondrial membrane potential is an early event of apoptotic cell death. The mitochon-

Figure 2 Staining of apoptotic bodies in MPPa PDT-treated HONE-1 cells. HONE-1 cells were pre-treated with MPPa (2 ␮M) for 24 h. The cells were stained with Hoechst 33258 at 16 h after light irradiation. HONE-1 cells (a); MPPa-treated HONE-1 cells (b); HONE-1 cells irradiated with 0.4 J/cm2 (c); or 1 J/cm2 (d); and MPPa-treated HONE-1 cells irradiated with 0.4 J/cm2 (e); or 1 J/cm2 (f). Arrow indicates the apoptotic cells.

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Figure 3 Confocal micrographs of HONE-1 cells stained with MPPa and organelle probes. HONE-1 cells were stained with MPPa (2 ␮M) for 24 h. The cells were then stained (a) with the Mitotracker (100 nM, 30 min); (b) Golgi body probe D3521 (2 ␮M, 20 min); and (c) Lysotracker (250 nM, 30 min). The localization of MPPa in HONE-1 cells is shown in red and the localization of organelle probes is shown in green. The yellow dots represent the combined confocal images of both MPPa and organelle probes.

drial localization of MPPa and the appearance of apoptotic bodies after MPPa PDT prompted us to examine the possible involvement of mitochondriamediated apoptotic cell death pathway after MPPa PDT treatment. HONE-1 cells were pre-treated with 2 ␮M MPPa and then irradiated with various light dose. Mitochondrial membrane potential was determined at 15 min after light irradiation. Noticeable change of mitochondrial membrane potential was not seen in control MPPa-treated HONE-1 cells (Fig. 5a). However, MPPa PDT-treated HONE1 cells underwent mitochondrial membrane depolarization, as judged from a leftward shift of the fluorescence curve in the fluorescence profile (Fig. 5b and c). It has been previously demonstrated that mitochondria trigger apoptotic cell death via the release of cytochrome c into cytoplasm. MPPa PDT-treated HONE-1 cells were then examined for cytochrome c release by immunofluorescence stain-

ing and the proportion of cells showing cytochrome c release was enumerated using flow cytometry as described previously [14]. The percentage of HONE1 cells showing the release of cytochrome c at 1 and 2 h after PDT was 39.3 and 98.9%, respectively (Fig. 6b and c). The presence of cytochrome c in the cytoplasm was also clearly seen in the confocal micrographs taken from cells at 2 h after treatment with MPPa PDT (Fig. 6e).

Detection MPPa PDT-induced ER stress To determine whether MPPa also exerts ER stress in the HONE-1 cells, Western blotting analysis was performed to analyze the expression of ER stress proteins. As shown, in Fig. 7, calnexin expression was clearly up-regulated at 2 h after PDT, while upregulation of Grp78 and Grp94 was also observed at 3—6 h after PDT.

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Figure 4 Image analysis of HONE-1 cells. HONE-1 cells were stained with MPPa and organelle probes as described in Fig. 3. Red dots represent the fluorescence images of MPPa, and the green dots represent the fluorescence images of the organelle probes. The fluorescence intensity profiles of MPPa (red lines) and the organelle probes (blue lines) are drawn along the red arrow in the confocal images. AU: arbitrary fluorescence units.

Caspase activation In response to apoptotic stimuli, caspases are activated and act as key mediators of programmed cell death. Caspase-9 is the key initiator caspase in the mitochondria-mediated apoptotic cell death pathway, while caspase-3 is the downstream execution effector caspase. The activities of caspase-9 (Fig. 8a) and caspase-3 (Fig. 8b) were then measured at 4, 8, and 12 h after PDT treatment. Both caspases-3 and -9 activity reached peak values at 8 h after PDT. To verify that the key caspases are involved in MPPa PDT-induced apoptosis, corresponding cell-permeable specific caspase inhibitors were added into the cell culture at 30 min before light irradiation. About 50% reduction of photocytotoxicity was seen in cells pre-treated with

the caspase-9 inhibitor Z-LEHD-FMK or caspase-3 inhibitor I, indicating that caspases-9 and -3 are involved in MPPa PDT-induced apoptotic cell death (Fig. 9).

Expression of cytokine mRNA in MPPa PDT-treated HONE-1 cells The expression profile of cytokines (e.g. IL-1, IL6, and TNF-␣) in NPC biopsies has recently been examined [16]. However, the expression of these cytokines by the tumour cells has not been examined. In this study, HONE-1 cells were treated with MPPa (2 ␮M) for 24 h, and the expression of various cytokine genes was examined at 2, 4, 8, and 12 h after light irradiation. As shown in Fig. 10, the

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Discussion

Figure 5 Mitochondrial membrane depolarization in HONE-1 cells after MPPa/PDT. HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then labeled with TMRE (150 nM) for 30 min before flow cytometric analysis. Collapse of membrane potential was determined at 15 min after PDT. (a) MPPa-treated NPC cells; (b and c) MPPa-treated NPC cells irradiated with a light dose of 0.4 and 1 J/cm2 , respectively. The spectral shift of the fluorescence curve to the left indicates mitochondrial membrane depolarization. Solid line represents untreated HONE-1 cells, dashed line represents MPPa PDT-treated cells.

expression of IL-6, TNF-␣, and MPIF-1 were induced starting from 2 h after PDT; MIP-␤, MPIF-2 started to be induced at 4 h, while IL-1␤ was up-regulated from 8 h after PDT. The expression of chemokine RANTES was not detected in both untreated or MPPa PDT-treated HONE-1 cells.

In the past few years, we have been focusing our research efforts on evaluating the efficacy of longer wavelength absorbing photosensitizers on NPC cells [14,17]. Our long-term goal is to identify a photosensitizer that may be used clinically for the treatment of NPC. Because of the light harvesting property, chlorophyll derivatives have recently been evaluated for their suitability for use as photosensitizers [2,6,10,18—23]. In the pyropheophorbide-a series, photosensitizing efficacy increases with the length of the carbon chain and pyropheophorbidea analogs are more active than the related chlorin e6 derivatives [24]. Apart from the direct photocytotoxic effect on tumour cells, MPPa PDT activates NF-kB and down regulates the expression of the adhesion molecules, ICAM-1 and VCAM-1, in endothelial cells [25]. We have previously demonstrated that the chlorophyll derivative MPPa has a strong absorption at 674 nm in cells and it is also a potent photosensitizer for lung carcinoma cells [11]. In a previous study, Matroule et al. demonstrated that MPPa localized to the cell membrane and lysosomes of the HCT-116 human colon carcinoma cell, and triggered colon cell apoptosis [10,26]. However, the components involved in triggering mitochondria-mediated colon cell death were not identified. In the present study, we extend our research to examine the mechanism of NPC cell death induced by MPPa. The primary finding of this study is that MPPa localizes in the mitochondria and Golgi body and triggers the mitochondriamediated apoptotic cell death pathway in the NPC cells via the release of chromosome c and the subsequent activation of caspase-9 and caspase-3. Furthermore, collapse of the mitochondrial membrane potential, an event preceding the release of cytochrome c, was seen in MPPa PDT-treated NPC cells for the first 15 min after light illumination. The results clearly indicated that triggering of the mitochondria-mediated apoptotic pathway is one of the mechanisms induced by MPPa PDT. In addition to mitochondria, we have previously demonstrated that the ER also plays a role in PDT-induced apoptotic cell death of NPC cells [14]. In this study, MPPa PDT also induced ER stress, as judged by the increase of three ER resident stress proteins calnexin, Grp78, and Grp94, in the NPC cells. The results support our previous observation that the ER may also play a role in PDT-induced apoptosis of NPC cells. PDT-induced inflammatory reaction has been implicated in the induction of a host immune response that contributes to tumour destruction [27,28]. PDT has been shown to induce the gene

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Figure 6 Flow cytometric analysis of cytochrome c release in MPPa PDT-treated HONE-1 cells. HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then irradiated (1 J/cm2 ). PDT-treated HONE-1 cells were fixed and stained with FITC-conjugated anti-cytochrome c antibody. (a) MPPa treated HONE-1 cells. (b and c) are MPPa PDTtreated HONE-1 cells at 1 h (b) and 2 h (c) after light irradiation, respectively. (d) Confocal images of control HONE-1 cells. (e) Confocal images of MPPa PDT (2 ␮M, 1 J/cm2 ) treated HONE-1 cells captured at 2 h after light irradiation. Solid line represents untreated HONE-1 cells, dashed line represents MPPa PDT-treated cells.

expression of pro-inflammatory cytokines (e.g. IL1, IL-6, TNF-␣) and cytokines (e.g. IL-10) involved in immune regulation. Early studies on NPC biopsies showed that NPC tissues expressed a wide range of cytokines [16]. Recently, Du et al. have demonstrated that IL-6 mRNA expression was increased in hypericin PDT-treated CNE-2 cells [29]. Expression of alpha-chemokines such as IL-8 by NPC

cells has also been examined [30]. However, the expression of beta-chemokines (CC chemokines), the chemokines preferentially involved in recruiting monocytes and certain subsets of T cells, by NPC cells has not been examined. In the present study, we extend our research to examine the expression of IL-1␤, IL-6, TNF-␣, and the beta-chemokines (MIP-1␤, RANTES, MPIF-1 and MPIF-2) in both the

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Figure 7 Western blot analysis of ER stress proteins expression. HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then irradiated (1 J/cm2 ) and cell lysates were prepared at 1—6 h after light irradiation. Each lane was load with equal amount of cellular proteins (20 ␮g/lane). Lane (a) HONE-1 cell alone; (b) MPPa treated HONE-1 cells; (c—f) MPPa PDT-treated HONE-1 cells at 1, 2, 3 and 6 h after light irradiation.

untreated and MPPa PDT-treated NPC cells. With the exception of RANTES, MPPa PDT was found to up-regulate the gene expression of IL-1␤, IL-6, TNF-␣, MIP-1␤, MPIF-1 and MPIF-2 in HONE-1 cells. The role of IL-6 in PDT-induced inflammation and tumour cell killing has recently been investigated. IL-6 expression has been implicated to play a role in increasing the sensitivity of lung carcinoma to PDT [31]. However, in the basal cell carcinoma (BCC) model, over-expression of IL-6 in BCC cells was found to reduce PDT-induced apoptosis [32]. In the present study, we found that MPPa PDT triggered both apoptosis and IL-6 expression in PDT-treated

Figure 8 Activation of caspase-9 and -3 by MPPa PDT. HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then irradiated at the light dose of 0.4 J/cm2 . Cell lysates were prepared at 4, 8, and 12 h after light illumination. The activity of caspase-9 (a) and caspase-3 (b) was then determined. Specificity of the enzyme activity was verified by including the corresponding specific caspase inhibitor in the reaction mixture as provided by the kit. Results represent mean ± S.D. of three replicates. AU: arbitrary unit.

Figure 9 Inhibition of MPPa PDT-induced cell death by caspase inhibitors. HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then treated with cell-permeable caspase-3 inhibitor I (2 ␮M; 30 min, Calbiochem; cat no. 235423) or caspase-9 inhibitor Z-LEHDFMK (20 ␮M; 30 min, ICN Pharmaceuticals, Inc. cat no. FK022) at 37 ◦ C. The cells were then irradiated and further incubated at 37 ◦ C in CO2 incubator for 24 h. Cytotoxicity was determined using the MTT cytotoxicity assay. Results represent mean ± S.D. of three replicates.

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tial, release of cytochrome c into the cytosol, and the induction of apoptotic cell death via the activation of caspase-9 and -3. Up-regulation of proinflammatory cytokine and beta-chemokine genes was associated with the induction of apoptosis. Our studies also lay the mechanistic groundwork for further development of MPPa PDT in the treatment of NPC.

Acknowledgments This work was supported by the Research Grant Council of Hong Kong (HKUST/CA03-04/2 and HKBU2052/02M) and HKBU (FRG/03-04/II-08).

References Figure 10 RT-PCR analysis of cytokine genes expression. HONE-1 cells were treated with MPPa (2 ␮M) for 24 h. The cells were then irradiated at the light dose of 0.4 J/cm2 and further incubated for 2—12 h at 37 ◦ C. RT-PCR products were separated on 2% agarose. M, 1 kb DNA marker; lane 1, untreated HONE-1 cells; lane 2, MPPa-treated HONE-1 cells; lanes 3, 4, 5, and 6, MPPa PDT-treated HONE-1 cells collected at 2, 4, 8, and 12 h after light irradiation, respectively.

NPC cells. The discrepancy on the effect of IL-6 in cellular sensitivity may vary depending on the tumour type studied. MPIF-1 and MPIF-2 are two novel CC chemokines [33]. MPIF-1, also known as CK␤8 or CCL23, is constitutively released by monocyte-derived dendritic cells [34]. MPIF-1 has been shown to have chemotactic activity on monocytes and dendritic cells [34—36]. MPIF-2, also known as eotaxin-2, is a potent chemotactic chemokine for eosinophil and myeloid progenitors [37,38]. In this study, MPPa PDT was found to up-regulate the expression of both MPIF-1 and MPIF-2 in HONE-1 cells. The results indicate that MPPa PDT-induced chemoattractants by NPC may contribute to infiltration of inflammatory cells into the irradiated tumours. The role of betachemokines produced by PDT-treated NPC cells in tumour eradication is still under investigation.

Conclusion MPPa is a semi-synthetic photosensitizer derived from chlorophyll a. Using human HONE-1 NPC cells, we have shown that mitochondria are the novel cellular target of MPPa PDT. MPPa PDT caused the collapse of mitochondrial membrane poten-

[1] Brown SB, Brown EA, Walker I. The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol 2004;5:497—508. [2] Nyman ES, Hynninen PH. Research advances in the use of tetrapyrrolic photosensitizers for photodynamic therapy. J Photochem Photobiol B 2004;73:1—28. [3] Oleinick NL, Morris RL, Belichenko I. The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 2002;1:1—21. [4] Ali SM, Olivo M. Mechanisms of action of phenanthroperylenequinones in photodynamic therapy (review). Int J Oncol 2003;22:1181—91. [5] Gorman SA, Brown SB, Griffiths J. An overview of synthetic approaches to porphyrin, phthalocyanine, and phenothiazine photosensitizers for photodynamic therapy. J Environ Pathol Toxicol Oncol 2006;25:71—108. [6] Piette J, Volanti C, Vantieghem A, Matroule JY, Habraken Y, Agostinis P. Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers. Biochem Pharmacol 2003;66:1651—9. [7] Villeneuve L. Ex vivo photodynamic purging in chronic myelogenous leukaemia and other neoplasias with rhodamine derivatives. Biotechnol Appl Biochem 1999;30(Part 1):1—17. [8] Lustig RA, Vogl TJ, Fromm D, et al. A multicenter Phase I safety study of intratumoral photoactivation of talaporfin sodium in patients with refractory solid tumors. Cancer 2003;98:1767—71. [9] Tsukagoshi S. Development of a novel photosensitizer, talaporfin sodium, for the photodynamic therapy (PDT). Gan To Kagaku Ryoho 2004;31:979—85 [in Japanese]. [10] Matroule JY, Carthy CM, Granville DJ, Jolois O, Hunt DW, Piette J. Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization. Oncogene 2001;20:4070—84. [11] Sun X, Leung WN. Photodynamic therapy with pyropheophorbide-a methyl ester in human lung carcinoma cancer cell: efficacy, localization and apoptosis. Photochem Photobiol 2002;75:644—51. [12] Xu CS, Leung AW. Photodynamic effects of pyropheophorbide-a methyl ester in nasopharyngeal carcinoma cells. Med Sci Monit 2006;12:BR257—62. [13] Glaser R, Zhang HY, Yao KT, et al. Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-

258

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

K.M. Li et al. Barr virus that were derived from nasopharyngeal carcinomas. Proc Natl Acad Sci USA 1989;86:9524—8. Mak NK, Li KM, Leung WN, et al. Involvement of both endoplasmic reticulum and mitochondria in photokilling of nasopharyngeal carcinoma cells by the photosensitizer ZnBC-AM. Biochem Pharmacol 2004;68:2387—96. Mak NK, Leung CY, Wei XY, et al. Inhibition of RANTES expression by indirubin in influenza virus-infected human bronchial epithelial cells. Biochem Pharmacol 2004;67:167—74. Huang YT, Sheen TS, Chen CL, et al. Profile of cytokine expression in nasopharyngeal carcinomas: a distinct expression of interleukin 1 in tumor and CD4+ T cells. Cancer Res 1999;59:1599—605. Mak NK, Kok TW, Wong RN, et al. Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells. J Biomed Sci 2003;10:418—29. Anderson TM, Dougherty TJ, Tan D, Sumlin A, Schlossin JM, Kanter PM. Photodynamic therapy for sarcoma pulmonary metastases: a preclinical toxicity study. Anticancer Res 2003;23:3713—8. Gollnick SO, Evans SS, Baumann H, et al. Role of cytokines in photodynamic therapy-induced local and systemic inflammation. Br J Cancer 2003;88:1772—9. Kelleher DK, Thews O, Scherz A, Salomon Y, Vaupel P. Combined hyperthermia and chlorophyll-based photodynamic therapy: tumour growth and metabolic microenvironment. Br J Cancer 2003;89:2333—9. Lee WY, Lim DS, Ko SH, et al. Photoactivation of pheophorbide a induces a mitochondrial-mediated apoptosis in Jurkat leukaemia cells. J Photochem Photobiol B 2004;75:119—26. Lim DS, Ko SH, Lee WY. Silkworm-pheophorbide alpha mediated photodynamic therapy against B16F10 pigmented melanoma. J Photochem Photobiol B 2004;74: 1—6. Snyder JW, Greco WR, Bellnier DA, Vaughan L, Henderson BW. Photodynamic therapy: a means to enhanced drug delivery to tumors. Cancer Res 2003;63:8126—31. Pandey RK, Sumlin AB, Constantine S, et al. Alkyl ether analogs of chlorophyll-a derivatives: Part 1. Synthesis, photophysical properties and photodynamic efficacy. Photochem Photobiol 1996;64:194—204. Volanti C, Gloire G, Vanderplasschen A, Jacobs N, Habraken Y, Piette J. Downregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy. Oncogene 2004;23:8649—58.

[26] Matroule JY, Bonizzi G, Morliere P, et al. Pyropheophorbidea methyl ester-mediated photosensitization activates transcription factor NF-kappaB through the interleukin1 receptor-dependent signaling pathway. J Biol Chem 1999;274:2988—3000. [27] Castano AP, Mroz P, Hamblin MR. Photodynamic therapy and anti-tumour immunity. Nat Rev Cancer 2006;6:535—45. [28] Dougherty TJ, Gomer CJ, Henderson BW, et al. Photodynamic therapy. J Natl Cancer Inst 1998;90:889—905. [29] Du H, Bay BH, Mahendran R, Olivo M. Hypericin-mediated photodynamic therapy elicits differential interleukin-6 response in nasopharyngeal cancer. Cancer Lett 2005. [30] Du H, Bay BH, Mahendran R, Olivo M. Endogenous expression of interleukin-8 and interleukin-10 in nasopharyngeal carcinoma cells and the effect of photodynamic therapy. Int J Mol Med 2002;10:73—6. [31] Usuda J, Okunaka T, Furukawa K, et al. Increased cytotoxic effects of photodynamic therapy in IL-6 gene transfected cells via enhanced apoptosis. Int J Cancer 2001;93:475—80. [32] Jee SH, Shen SC, Chiu HC, Tsai WL, Kuo ML. Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency. Oncogene 2001;20:198—208. [33] Patel VP, Kreider BL, Li Y, et al. Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors. J Exp Med 1997;185:1163—72. [34] Nardelli B, Morahan DK, Bong GW, Semenuk MA, Kreider BL, Garotta G. Dendritic cells and MPIF-1: chemotactic activity and inhibition of endogenous chemokine production by IFNgamma and CD40 ligation. J Leukoc Biol 1999;65:822—8. [35] Forssmann U, Delgado MB, Uguccioni M, Loetscher P, Garotta G, Baggiolini M. CKbeta8 a novel CC chemokine that predominantly acts on monocytes. FEBS Lett 1997;408: 211—6. [36] Youn BS, Zhang SM, Broxmeyer HE, et al. Characterization of CKbeta8 and CKbeta8-1: two alternatively spliced forms of human beta-chemokine, chemoattractants for neutrophils, monocytes, and lymphocytes, and potent agonists at CC chemokine receptor 1. Blood 1998;91:3118—26. [37] Grzegorzewski KJ, Yao XT, Kreider B, et al. Analysis of eosinophils and myeloid progenitor responses to modified forms of MPIF-2. Cytokine 2001;13:209—19. [38] White JR, Imburgia C, Dul E, et al. Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils. J Leukoc Biol 1997;62:667—75.