Cancer Letters 242 (2006) 112–119 www.elsevier.com/locate/canlet
Photodynamic effects on nasopharyngeal carcinoma (NPC) cells with 5-aminolevulinic acid or its hexyl ester R.W.K. Wu a,b, E.S.M. Chu b, C.M.N. Yow b, J.Y. Chen a,* a
Department of Physics, State Key Laboratory for Advanced Photonic Materials and devices, Fudan University, Shanghai, China b Biomedical Science Section, School of Nursing, Hong Kong Polytechnic University, Hong Kong, People’s Republic of China Received 4 October 2005; accepted 29 October 2005
Abstract Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Hong Kong and southern China. To explore a new modality of NPC treatment, 5-aminolevulinic acid (ALA) or its hexyl ester (ALA-H) mediated photodynamic therapy (PDT) was studied in vitro. The results show that NPC cells are sensitive to both ALA and ALA-H mediated PDT. However, ALA-H PDT is much more effective at cell inactivation than ALA-PDT, due to a higher efficiency of ALA-H on producing endogenous protoporphyrin (PpIX) in cells. Both apoptosis and necrosis are involved in cell death, but apoptosis plays a major role under the short time incubation of drugs. ALA and ALA-H mediated PDT not only destroy the cells directly, but also inhibit the expression of matrix metalloproteinase-2 (MMP2) in cells, a maker for tumor metastasis. The ALA-H shows promising PDT results on NPC in vitro; therefore it is worth investigating further in vivo for NPC treatment. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Nasopharyngeal carcinoma; 5-Aminolevulinic acid hexyl ester; Photodynamic therapy; Metastasis
1. Introduction Nasopharyngeal carcinoma (NPC) is malignant squamous cell carcinoma of the head and neck region that has the highest prevalence in Hong Kong and South China. The NPC incidence of South China was reported as 30–50/100,000, which is 25 times higher than that in Europe and USA . The common treatments are the radiotherapy and chemotherapy, but the recurrent rate is high . Photodynamic therapy (PDT) as a promising modality for cancer treatment had treated thousands tumor patients successfully worldwide [3,4]. The PDT clinical treatment of NPC has been carried out also in China with the 1st generation photosensitizer of * Corresponding author. Tel.: C86 21 65642366; fax: C86 21 65104949. E-mail address: [email protected]
hematoporphyrin derivative (HPD), and the result was encouraging [5,6]. For exploring a better PDT modality on NPC treatment, in the past years we studied several photosensitizers in vitro and found that the Temoporfin (mTHPC) is the best one on NPC photo-inactivation [7,8]. However, both HPD and mTHPC cause a prolonged skin photo-toxicity that makes their application limited. The search for an ideal photosensitizer with low skin photo-toxicity is still going on . Recently the considerable interest has been directed towards developing new PDT regimens that rely on an endogenously synthesized sensitizer. A pro-drug, 5-aminolevulinic acid (ALA) has been focused in this field . ALA is a precursor of several porphyrin intermediates particularly protoporphyrin IX (PpIX) in the heme biosynthetic pathway, and the PpIX is a potent sensitizer. If extra exogenous ALA is introduced into cells, endogenous protoporphyrin (PpIX) formation will be
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.10.048
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enhanced and accumulated in cells. Upon activation by light, the accumulated PpIX initiates the photodynamic reaction, called ALA-PDT. ALA-PDT has several outstanding characteristics. Firstly, ALA-derived PpIX can be cleared from the body within 24–48 h after systemic administration. This would reduce and avoid the risk of prolonged skin phototoxicity . Secondly, the drug delivery is flexible. It has been reported that oral intake of ALA gives similar bio-distribution kinetics to intravenous administration , and finally, topical application has been successfully used in clinic for skin cancers . Due to these advantages the pioneer work has focused on testing ALA-PDT on NPC in vitro  as well in vivo  with promising results. However, so far the data of ALA-PDT effects on NPC are still not so sufficient. More recently, the ALA hexyl-ester (ALA-H) has been found to be much more effective than ALA at photosensitizing several cancer cell lines . The aim of the present study was to compare PpIX production, photocytotoxicity and cell death pathways between ALA and ALA-H in NPC/CNE2 cell line. The PDT effect on the expression of matrix metalloproteinase-2 (MMP2) in NPC cells was also studied. 2. Materials and methods 2.1. Cell line The nasopharyngeal carcinoma (NPC/CNE2) cell line was a poorly differentiated squamous cell carcinoma from a 68year-old Chinese male . Cells were routinely cultured in a RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and antibiotics PSN (50 IU/ml penicillin, 50 mg/ml streptomycin and 100 mg/ml neomycin) at 37 8C in a humidified 5% CO2 incubator. 2.2. Chemicals Both ALA and ALA-H (both are hydrochoride salts) were provided by PhotoCure ASA (Oslo, Norway). Stock solutions were prepared in Dulbecco’s PBS (GibcoBRL, Life Technologies) at a concentration of 10 mM for ALA and 1 mM for ALA-H, and stored at 4 8C in the dark in a few days. Serumfree RPMI medium was used to further dilute the stock solutions to the desired concentrations for experiments. 2.3. Cytotoxicity assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was used to assess the ALA-PDT phototoxic effect on NPC cells . MTT assay was carried out in 96-well flat bottom plates. 100 mL NPC cell suspension (3!104) were added to each well and incubated
for 24 h. Different concentrations of the drug were added to each well and incubated at 37 8C in a 5% CO2 incubator for 4 h. Cells were washed twice with PBS and then fresh medium was added before being irradiated with different light doses from a 400 W quartz-halogen lamp with a heat isolation filter and a 600 nm long-pass filter. The spectral intensity of light source was basically flat from 600 to 800 nm and the total intensity was measured to be at 14 mW/cm2 using a power meter . After irradiation, cells were incubated for 24 h and cell survival was determined by MTT assay using iEMS Analyzer (Lab-system). Appropriate controls were included in each experiment. All results were presented as the meanGSD from three to six independent experiments with six wells in each. 2.4. Detection of PpIX accumulation in cells by flow cytometric analysis About 3!105 cells were seeded in 60 mm culture dishes (Corning) and incubated at 37 8C in a 5% CO2 humidified incubator for 24 h. ALA or ALA-H was then added and incubated for different times. After incubation, the cells were trypsinized with 0.25% trypsin–EDTA, washed and resuspended in 0.5 ml DPBS. All procedures were performed in the dark. PpIX production in the cells was measured by flow cytometry (EPICS, Coulter Elite), in which the excitation was 488 nm from an argon ion laser and the fluorescence signal was selected with a 600 nm long-pass filter . About 20,000 cells were measured for each sample. Background fluorescence from control cells (without drug) was subtracted in the measurement. The fluorescence intensities in different cell populations were quantified. Results were expressed as mean G SD from three to six independent experiments. 2.5. Intracellular localization of PpIX in NPC cells Cells were incubated with different concentrations of ALA or ALA-H, respectively. The fluorescence images of cells were captured using a laser excitation confocal microscope (PerkinElmer, Ultraview LCI). The excitation was at 488 nm from an attached argon-krypton laser, and a 590 nm long-pass filter was used for acquiring the fluorescence image. This confocal microscope provides a good resolution on the z-axis of about 0.3 mm. The imaging measurements were repeated at least six times, and hundreds cells were observed. 2.6. The examination of apoptotic/necrotic cells in fluorescence microscopy with AO/EB staining Apoptotic cells post-PDT were detected in a fluorescence microscope (Nikon Eclipse E600) with acridine orange (AO) /ethidium bromide (EB) staining. The AO/EB staining is the popular method for apoptotic and necrotic assay . The EB stains nuclei only when the nuclear membrane is damaged, and emits red-orange fluorescence detected in red channel of the microscope with the 590 nm long-pass filter. The AO can
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penetrate through the nuclear membrane to stain the nuclei, and emits the green fluorescence imaged in green channel (520–550 nm band-pass filter). In apoptotic cells, the green granules called apoptotic body appear in the nuclei, due to the chromatin condensation and nuclear fragmentation. The cells were incubated with ALA (1 mM) or ALA-H (0.1 mM) for 4 h, respectively and then irradiated with the light dose of 2 J/cm2. After light irradiation, the cells were stained with AO/EB mixture (2 mg/ml AO (Sigma) and 2 mg/ml EB (Sigma)) for 15 min. These stained cells were examined in the fluorescence microscope in both red channel and green channel. The excitation is a mercury lamp with filter of 400–440 nm. A 510 nm dichromic filter was also used in microscope to separate the excitation and emission. The measurements were carried out for three to six times.
death is both drug concentration and irradiation dose dependent. But when the incubation concentration of ALA is over 0.5 mM and that of ALA-H is over 0.01 mM, a saturating tendency of the cytotoxicity is shown. Furthermore, ALA-H is much more effective than ALA at inactivation of cells. To achieve an 80% death rate (LD80), 0.75 mM ALA and 6 J/cm2 light dose were required, while only 0.03 mM ALA-H and 4 J/cm2 light dose were needed. In terms of overall photodynamic dose (drug dose!light dose), ALA-H is about 40 times more potent than ALA in cell inactivation. 3.2. Kinetics of PpIX accumulation in cells
2.7. The apoptotic/necrotic cells measured by light scattering in Flow cytometry Cell shrinkage usually occurs during apoptosis and results in a decrease in forward light-scatter in the measurement of flow cytometry . It is thus possible to utilize the change of light-scattering properties to distinguish apoptotic cells from necrotic and alive cells by gating procedures of flow cytometric analysis . Cell samples for such study were incubated with ALA (1 mM) or ALA-H (0.03 mM) for desirable times, and then irradiated by the light with 4 J/cm2 dose. After 8 h followed PDT, these cell samples were measured in flow cytometry, and about 20,000 cells of each sample were analysed using the proper gating procedure based on the signals of forward light scatter versus that of side light scatter . All results were presented as the meanGSD from three to six independent experiments.
In both ALA-PDT and ALA-H-PDT, the PpIX formation in cells is a key factor. The PpIX cellular accumulation was measured with different incubation times of drug. Fig. 2 summarizes the kinetics of cellular PpIX accumulation. The PpIX accumulation tendency of two curves (ALA and ALA-H incubated cells) is similar. The cellular PpIX content increases linearly with drug incubation time in early stage, and then
2.8. Determination of matrix metalloproteinase-2 in cells The metastasic protein–matrix metalloproteinase-2 (MMP2) is believed as the typical marker of metastasis ability for some cancer cells . The effects of ALA-PDT or ALA-H-PDT on MMP2 expression of the NPC cells were detected with MMP2-antibody (Oncogene, USA). After PDT these cell dishes (containing 106 cells in each dish) were added with serum-free RPMI medium (2 ml) for another 24 h to harvest the MMP2 in medium. The MMP2-antibody was used to quantify the amount of the MMP2 in each sample by measuring the OD value at 590 nm in ELISA.
3. Results 3.1. Phototoxicity of NPC cells mediated by ALA-PDT and ALA-H-PDT Fig. 1 shows the results of MTT cytotoxic assay on cells after ALA or ALA-H mediated PDT. The cell
Fig. 1. Photo-cytotoxicity of ALA and ALA hexyl ester on NPC cells. Cells were incubated with different ALA or ALA-H concentrations for 4 h, and then irradiated with various light doses. The death rate was measured by MTT assay. Error bars (SD) were from 3 to 6 independent experiments. (a) ALA; (b) ALA-H.
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Fig. 2. Kinetics of cellular PpIX accumulation with drug incubation times. The fluorescence intensity represents the cellular PpIX content (relative). Cells were incubated with ALA (1 mM) or ALA-H (0.03 mM) for different times. Error bars (SD) were obtained from 3 to 6 independent experiments.
Fig. 4b and c, indicating that the early stage apoptosis occurred. The EB dyes stain the nuclei only when the nuclear membrane is damaged. During the necrosis or later stage apoptosis, the nuclear membranes have been broken, thus the stained nuclei can be detected by recording the EB signals in red channel. The Fig. 4d and e shows the images of cells 2 h after ALA-PDT and ALA-H-PDT measured in red channel, demonstrating that both apoptosis (nuclei condensation with apoptotic bodies) and necrosis (without apoptotic bodies) happened. The apoptotic and necrotic cells were investigated further by flow-cytometry with cell-size dependent light scattering measurements. At 8 h after PDT, the necrotic cells have broken into debris, resulting in a decrease in both forward scattering light (FSC) and side scattering light (SSC). While for the apoptotic cells,
saturates around 15–20 h. Fig. 2 also demonstrates that the cellular PpIX content in 1 mM ALA incubated cells is similar to that in 0.03 mM ALA-H incubated cells, reflecting that ALA-H is more efficient than ALA on inducing PpIX production. And these results may explain why ALA-H is more effective than ALA on photo-inactivation of cells. 3.3. Intracellular localization of PpIX After being incubated with ALA or ALA-H, the fluorescence images of these cells were acquired in the confocal microscope, as shown in Fig. 3. The diffuse cytoplasmic distribution is the typical pattern of PpIX intracellular localization. There is no obvious difference in the images between the ALA incubated cells and ALA-H incubated cells. Interestingly, no PpIX enters the nuclear region even after 24 h incubation with ALA or ALA-H. 3.4. The mechanism of cell death mediated by ALA-PDT and ALA-H-PDT PDT can damage the cells in vitro via apoptosis and the necrosis . In the present study, apoptosis and necrosis were qualitatively examined with fluorescence microscope using AO/EB staining. Fig. 4 shows the images of AO/EB stained cells. The apoptotic bodies resulting from the chromatin condensation were clearly seen shortly after ALA-PDT and ALA-H-PDT in
Fig. 3. Intracellular localization of PpIX in NPC cells. Fluorescence images were acquired from confocal imaging system with LP 590 nm filter set. Excitation: 488 nm from ArC laser. Cells were incubated with ALA (2 mM) or ALA hexyl ester (0.1 mM) for 11 h. (a) ALA loaded cells; (b) ALA-H loaded cells.
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Fig. 4. The morphological examination of apoptotic and necrotic cells with (AO/EB) staining. The AO stained nuclei were detected in green channel with 520–550 nm band-pass filter, as in (a) control cells; (b) 1 h after PDT treatment (1 mM ALA incubation and 2 J/cm2 irradiation); (c) half hour post-PDT with 0.01 mM ALA-H incubation and 2 J/cm2 irradiation. The gray dotted arrows show the early stage apoptotic cells in (b) and (c). The EB stain nuclei were measured in red channel with 590 nm long-pass filter in (d) 2 h after ALA (1 mM, 2 J/cm2) treatment; (e) 2 h after ALA-H (0.01 mM, 2 J/cm2) treatment. The white arrows point out the necrotic cells in (d) and (e).
the cell shrinkage mainly lowered the FSC signals, giving rise to a low ratio of FSC/SSC. Using a proper gating based on FSC versus SSC, the apoptotic cells and necrotic cells could be quantitatively measured in the cell samples . As shown in Fig. 5, the gating lines divide the whole area into several regions, in which the F2 represents the fraction of living cells, the F1 for apoptotic and F3 for necrotic fraction. The control cells were measured and showed in Fig. 5a with the alive cells of 97% under this gating condition. Fig. 5b and c gives the typical diagrams of the cell samples being incubated with ALA-H (0.03 mM) for 4 and 20 h, respectively and irradiated with equal 4 J/cm2 light dose. It can be seen that after ALA-H-PDT all the three kinds of cells (apoptotic, necrotic and living cells) exist with different fractions. The similar results of flow cytometry are found in cells treated with ALA-PDT. The statistical data are summarized in Table 1, that are in agreement with results of AO/EB staining, further confirming that both apoptosis and necrosis are involved in ALA and ALA-H mediated PDT of NPC cells. 3.5. The PDT effect on MMP2 expression Fig. 6 shows the PDT effects on MMP2 expression in NPC cells. Both ALA-PDT and ALA-H-PDT can decrease the MMP2 content in treated cells. When cells were treated with ALA-PDT at the doses of
0.25 mM!4 J/cm2 and 0.5 mM!4 J/cm 2, which correspond to the LD25 and LD40 death rates of cells, a significant lower expression of MMP2 content has been found (one-way ANOVA, p!0.01). Similarly ALA-H-PDT treatments (LD50 and LD-80) lowered the MMP2 contents too (p!0.01), indicating that PDT suppressed the MMP2 expression in NPC cells. 4. Discussion Among ALA derivatives, esterified ALA was reported to enhance PpIX production . In the esterified family of ALA, the ALA-H showed a better PDT effect than others on human adenocarcinoma cell lines  and human cervical carcinoma cells . Here, the ALA-H was selected to compare with ALA on photosensitization of NPC cells. Our results confirm that ALA-H has a higher efficacy than ALA on photoinactivation of NPC cells in vitro, thus suggesting that ALA-H may have potential for PDT application of NPC and the in vivo work should be investigated further. To achieve LD80 death rate in NPC cells, only 0.75 mM ALA incubation (4 h) and 6 J/cm2 irradiation were needed, reflecting that the NPC cells are more sensitive to ALA-PDT than other types of tumor cell lines such as cervical carcinoma cell lines (C-33A and CaSki) , H29 colorectal adenocarcinoma cells , L1210 leukemia cells , C6 glioma cells  and radiation-induced fibrosarcoma (RIF) cells .
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the plasma membrane entering the cells. While the ALA is a hydrophilic compound, its permeability of plasma membrane is limited. As shown in Table 1, both apoptosis and necrosis occur in ALA and ALA-H mediated PDT of NPC cells. The fractions of apoptotic cells and necrotic cells are drug incubation time related. At a short time incubation (4 h), the apoptosis is most seen. While for a long time incubation (20 h) the necrosis becomes pronounced. This may be due to the fact that in a short time incubation, PpIX is relatively concentrated in mitochondria, thus resulting in the mitochondriainitiated apoptosis in cells. When the incubation is prolonged, more PpIX molecules diffuse into the whole cytoplasm leading to necrosis of cells. The expression of MMP2 is associated with the high potential of metastasis in several human carcinomas . Especially, it has been reported recently that the MMP2 level is correlated to the survival of the patients with poor differentiated NPC . Therefore, the Table 1 Statistic analysis of the apoptotic and necrotic cells after ALA-PDT (1 mM!4 J/cm2) or ALA-H-PDT (0.03 mM!4 J/cm2), by scattering measurements in flow cytometry (data based on 3–4 independent experiments) Incubation time (h)
Fig. 5. The apoptotic/necrotic cells quantitated in scattergrams of flow cytometry. (a) Control cells; (b) cells after treatment of 4 h incubation of ALA-H (0.03 mM) and 4 J/cm2 irradiation; (c) cells after 20 h incubation of ALA-H (0.03 mM) and 4 J/cm2 irradiation.
However the NPC cells are even more sensitive to ALA-H-PDT. The overall dose of ALA-H-PDT for reaching LD80 in NPC cells is much lower than that in cervical carcinoma cells , indicating that the NPC cells even better suit for ALA-H mediated PDT in vitro. In this comparative study, the ALA and ALA-H show very similar patterns of PpIX intracellular localization as well as the kinetics of PpIX accumulation in cells. The main difference is that the ALA-H is much more efficient at inducing PpIX production in NPC cells than ALA, resulting in the much higher efficiency of ALA-H on the cell inactivation. Because the ALA-H molecules are more lipophilic, they are easy to penetrate through
ALA-H 4 20 ALA 4 20
Apoptotic cell (F1, %)
Necrotic cell (F3, %)
Fig. 6. PDT effects on MMP2 expression. When cells were incubated with ALA or ALA-H and then irradiated with 4 J/cm2 light dose, the MMP2 level in each cell sample was measured with the MMP2antibody 24 h after PDT. Results are meanGSD of three independent experiments.
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MMP2 level was studied to evaluate metastasis potential post-PDT in NPC cells. Both ALA and ALA-H mediated PDT can significantly inhibit the MMP2 expression as shown in Fig. 6. After ALA-PDT with the doses of LD25 and LD40, the MMP2 contents were 7.5 and 2.5 ng/ml, respectively, in contrast with the 15 ng/ml of control cells, reflecting that 50 and 83% MMP2 contents have been suppressed. Considering the death rates of cells were LD25 and LD40, the results indicate that the MMP2 expression has been inhibited in these left alive cells. The similar expression was found in ALA-H-PDT treated cells. With the PDT doses of LD50 and LD80, only 33 and 7% MMP2 contents remained, respectively, demonstrating that PDT may have potential for inhibiting tumor metastasis.
Acknowledgements We thank the Research Grants Council of Hong Kong for the support of this work.
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