ALA-Porphyrin Science
Tumor Suppressor Protein p53-dependent Cell Death
Induced by 5-Aminolevulinic Acid (ALA)-based Photodynamic
Sensitization of Cancer cells in Vitro
Yuichiro Hagiyaa†, Yoshio Endob†, Yutaka Yonemurac, Ichiro Okuraa, and Shun-ichiro Oguraa*aTokyo Institute of Technology , 4259 Nagatsuta-cho, Midori-ku, Yokohama, Japan. Fax: +81-45-924-5845; Tel: +81-045-924-5845;
E-mail:hagiya.y.aa@m.titech.ac.jp (Y.H.), sogura@bio.titech.ac.jp (S.O.)b
Kanazawa University, Kakuma-machi, Kanazawa, Japan. Tel: +81-76-264-6775; E-mail: yendo2@staff.kanazawa-u.ac.jpc

NPO Organization to support Peritoneal Dissemination Treat.1-26, Haruki-Moto-macho, Kishiwada,Osaka, Japan. Tel: +81-72-433-2131; E-mail:y.yonemura@coda.ocn.ne.jp† The first and second authors are equally contributed to this work.

Received 30th November 2011, Accepted 8th December 2011

Summary
Recently, photodynamic therapy using 5-aminolevulinic acid (ALA-PDT) is widely used incancer therapy, owing to the tumor-specific accumulation of photosensitizing protoporphyrin IX(PpIX) after administration of ALA. In the present study, by focusing on ALA-based photodynamic
challenge (ALA-PDC) with 640 nm of LED, we examined the potential involvement of
p53-mediated apoptosis in 3 human gastric cancer cell lines. We found the cytotoxic effects of
ALA-PDC are dependent on both the ALA concentration and the irradiation dose of LED. Typicalmorphological changes of apoptotic and/or necrotic cell death caused by ALA-PDC were observedby propidium iodide and Hoechst 33258 staining. At the higher concentrations of ALA, necrotic cells
were increased whereas apoptotic cells were decreased. Among three cell lines tested, we found that
MKN45 cells which possess wild-type p53 gene are highly sensitive to ALA-PDC. However,MKN45 cells after treatment with p53 specific inhibitor pifithrin-α showed significantly decreased
sensitivity to ALA-PDC. Immunoblotting analysis confirmed that Ser15 in p53 was phosphorylated
following ALA-PDC in MKN45 cells, suggesting p53 protein stabilization and transcriptional
up-regulation of p53 target genes. Conversely the photosensitivities of KKLS and MKN28 cells
which harbor mutant p53 gene were little affected by pifithrin-α. Thus, we propose that the genestatus of p53 is potential to predict the patient’s response to ALA–PDT and the patient who has
wild-type p53 gene would be sensitive for ALA–PDT.
Keywords:
Photodynamic therapy, p53, 5-aminolevulinic acid, apoptosis, necrosis, pifithrin-α
Introduction
Photodynamic therapy (PDT) is an advantageous anti-tumor treatment that utilizes thegeneration of reactive oxygen species in tumor tissue [1-3]. 5-Aminolevulinic acid (ALA) is
recently used for PDT, owing to the tumor-specific accumulation of photosensitizing
This journal is copyright The ALA and Porphyrin Research Society 2012
This journal is copyright The ALA and Porphyrin Research Society 2012

ALA-Porphyrin Science, 2011, 1, 21–29 | 21

ALA-Porphyrin Science, 2012, 1, 23 ─ 31

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protoporphyrin IX (PpIX) after administration of ALA [3; 4]. As ALA can be applicable orally or
topically, ALA administration has become a widely acceptable and popular procedure, particularly
for the treatment of skin cancer [4]. Accumulation of ALA in cancer cells rapidly increases theintracellular porphyrin levels preferentially in neoplastic regions. ALA-induced intracellularaccumulation of protoporphyrin IX (PpIX) is also used for fluorescence diagnosis of premalignant
and malignant diseases in dermatology, urology, neurosurgery, otorhinolaryngology, gynecology,
and gastroenterology [4; 5].
p53 plays the central role of tumor suppressor protein to restrict tumor development by serving
as a sensor of cellular stress, such as DNA damage and oxidative stress [6; 7]. p53 is also involvedin triggering apoptosis. p53 protein is composed of a core DNA-binding domain and several
regulatory domains such as trans-activation domain, proline-rich domain, tetramerization domain,and carboxy terminal regulatory domain. Over 50% of human cancers reportedly possess p53
mutation. Most of p53 mutations are missense mutations found in DNA-binding domain known as
hot spot, showing the ablation of apoptosis. In this regard, it is of importance that loss of function of
wild-type p53 decreases the efficacy of anti-cancer treatments such as radiation therapy andchemotherapy [8; 9]. Hitherto, the signal transduction involved in PDT-mediated cell death using
several types of photosensitizers has been reportedly investigated so as to make the consideration to
the efficacy of PDT [10-15]. Fisher et al reported that p53 expression does not directly modulate
tumor cell sensitivity to Photofrin-PDT in either apoptosis-responsive (LS513) or nonresponsive(MCF-7) cells. Lee et al also suggested that the gene status of p53 may not be important in
hypericin-PDT-mediated cell killing or induction of apoptosis [11; 15].
In this work, we investigated the involvement of p53 on ALA-based photodynamic challenge(ALA-PDC) with 640 nm of LED irradiation. We examined the potential involvement of
p53-mediated apoptosis in ALA-PDT using a p53 specific inhibitor pifithrin-α [16] showing
pifithrin-α inhibited p53-mediated apoptosis. The present study provides evidence that the status ofp53 gene in cancer cells is practical and useful biomarkers for predicting the photo-sensitivity to
ALA-PDT.
Experimentals
Biochemicals
5-Aminolevulinic acid (ALA) hydrochloride was purchased from COSMO OIL CO., LTD.
(Tokyo, Japan). Pifithrin-α was purchased from ALEXIS (Lausen, Switzerland). MTT reagent,propidium iodide (PI), and Hoechst 33258 were purchased from Sigma–Aldrich (St. Louis, MO).
RPMI-1640 medium and protease inhibitor cocktail were obtained from Nacalai Tesque (Kyoto,
Japan). Penicillin–streptomycin and FBS were purchased from Invitrogen (Carlsbad, CA). All otherchemicals used were of analytical grade.
Cells and cell culture
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The human gastric cancer cell lines KKLS [17] were kindly provided by Dr. Mai of Cancer
Research Institute of Kanazawa University (Kanazawa, Japan). MKN45 and MKN28 cells werekindly provided by Dr. Suzuki (Fukushima Medical College, Fukushima, Japan). Cells weremaintained at 37°C under 5% CO2 gas in RPMI-1640 medium supplemented with 10% (v/v)
heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.
Detection of non-synonymous p53 mutation in human gastric cancer cells
To detect non-synonymous mutation of p53 in MKN45, MKN28, KKLS cells, the coding
region of p53 cDNA (NM_000546) were amplified by RT-PCR using the sense primer p53-37seq:
5’-TTG CCG TCC CAA GCA ATG GAT GAT TTG ATG CTG TC-3’ and the antisense primer
p53-2seq: 5’-GCG GAG ATT CTC TTC CTC TGT GCG CCG GTC TCT CCC A-3’. The sequenceof most common amino acid residues altered region in cancer known as hotspots in p53 wasdetermined by direct sequence method using the sense primer p53-37seq: 5’-TTG CCG TCC CAAGCA ATG GAT GAT TTG ATG CTG TC-3’, p53-seq5: 5’-GAT GAA GCT CCC AGA ATG CCA
GAG GCT GCT-3’, p53-1seq: 5’-ACC TGC CCT GTG CAG CTG TGG GTT GAT TCC ACA-3’,
and p53-seq4: 5’-TTG CGT GTG GAG TAT TTG GAT GAC AGA AAC ACT TTT-3’, then theanti-sense primer p53-44seq: 5’-GCA AGT CAC AGA CTT GGC TGT CCC AGA ATG CAA G-3’,
p53-seq6: 5’-TCT CCC AGG ACA GGC ACA AAC ATG CA-3’, and p53-2seq: 5’-GCG GAG ATTCTC TTC CTC TGT GCG CCG GTC TCT CCC A-3’. We confirmed the p53 status of those celllines; MKN45 cells possess wild-type p53 whereas KKLS cells (Gly245Asp) and MKN28 cells(Ile251Leu) possess non-synonymous p53 mutation.
Incubation of human gastric cancer cells with ALA and exposure to light-emitting diode (LED)irradiation
Cells were seeded in a 96-well plate (1  104 cells/well) and cultured at 37°C under 5% CO2
gas for 24 h. ALA was added to the culture medium at increased concentrations, and incubation
continued in the dark for 4 h. Thereafter, cells were exposed to LED irradiation for designated time
(630 nm, 3.6 mW/cm2) by placing the cell culture plate below an LED irradiation unit provided by
SBI ALA PROMO (Tokyo, Japan). Cells were further incubated in the dark for 24 h, and cell
viability measured by the MTT assay as described previously [18]. The absorbance of formazan (ametabolite of MTT) in the resulting solution was measured photometrically at a test wavelength of
570 nm and a reference wavelength of 655 nm in a Microplate reader Model 550 (Bio-Rad
Laboratories, Hercules, CA). IC50 values were calculated from dose–response curves (i.e., cell
survival vs. ALA concentration) obtained in duplicate experiments.
In this context, in order to investigate classification of cell death - i.e. apoptosis and/or necrosis,
MKN45 cells incubated for 24 h following photodynamic treatment were stained with 5 μM of PI
and Hoechst 33258 in culture medium for 1 h. Fluorescence of PI or Hoechst 33258 was detectedwith a fluorescence microscope (IX70/FLUOVIEW; Olympus, Tokyo, Japan).
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Western blot analysis
For immunoblot analysis, cell lysates were prepared as described previously [18]. Samples
were first treated with the SDS-PAGE sample buffer solution. Thereafter, sample proteins wereseparated electrophoretically by using a 12% polyacrylamide gel and electroblotted onto an
Immobilon-P PVDF membrane (Millipore Corp., MA). The membrane was incubated in a blocking
solution containing 5% (w/v) skim milk in TTBS [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05%(v/v) Tween 20] at room temperature for an hour.
We used a polyclonal antibody specific to the phosphorylated p53 at Ser15 (Cell signaling
technology, Boston, MA; 1:1000 dilution), or monoclonal antibody against rabbit muscle GAPDH(American Research Products, Belmont, MA; 1:1000 dilution) as the first antibody, depending on
the specific purpose of the immunoblot analysis. For the second antibody, we used anti-mouse IgG
horseradish peroxidase (HRP)-conjugated antibody (Cell Signaling Technology, Beverly, MA) andanti-rabbit IgG HRP conjugates (Santa Cruz Biotechnology) at 1:3000 dilution.
HRP-dependent luminescence was developed with Western Lightning Chemiluminescent
Reagent Plus (PerkinElmer Life and Analytical Sciences, Waltham, MA) and detected with a
Lumino Imaging Analyzer LAS-4000mini (GE Healthcare UK, Amersham Place, England).
Results and Discussion
Photosensitivities of human gastric cancer cells are dependent on ALA and irradiation dose.
In the present study, in order to evaluate the cellular photosensitivity, we first treated cells in thepresence of ALA for 4 h in the dark. Cells were then exposed to LED light (630 nm, 3.6 mW/cm2)
for 0, 0.5, 1, 2.5, 5, 10 min, and the cell viability was analyzed using MTT assay. Figure 1
demonstrate the LED irradiation and ALA dose-responses of MKN45, KKLS

Figure 1. The dose responses of human gastric cancer cells to photodynamic sensitization following
incubation with ALA. The cellular sensitivity by ALA-PDC is dependent on the ALA- andirradiation-dose. (A) MKN45 cells (wild-type p53) (B) KKLS cells (mutant p53; Gly245Asp) (C)MKN28 cells (mutant p53; Ile251Leu).
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, and MKN28 cells to ALA-PDC. Each cell lines exhibited photosensitivity depending on ALA
concentration and irradiation energy of LED light. The cell viability of the control cells was notaffected by irradiation with the LED light in the absence of ALA (data not shown). The IC50 values
for 10 min (2160 mJ/cm2) of MKN45, KKLS, MKN28 cells were calculated to be 100, 700, and1000 μM for ALA, respectively. Compared with MKN45 posseing wild-type p53 gene, KKLS and
MKN28 harboring mutant p53 gene showed lower sensitivity to ALA-PDC. These results suggestthat gene status of p53 would determine the efficacy of ALA-PDT.
ALA-PDC induced apoptosis in MKN45 cells associated with chromatin condensation byphotosensitization
Multiple signaling cascades are concomitantly activated in cancer cells exposed to thephotodynamic stress. Depending on the subcellular localization of cytotoxic ROS, those signals aretransduced into adaptive or cell death responses [19]. To address the question of whether apoptosis
occurs in the case of ALA-PDC, we investigated the morphological change of MKN45 cells causedby photosensitization following incubation with 100 µM ALA for 4 h. Using PI and Hoechst33258
staining, typical morphological change of apoptotic and/or necrotic cell death caused by ALA-PDC
were observed. Fluorescence staining with Hoechst 33258 (blue) revealed the ALA-PDC induces
chromatin condensation leading to apoptosis (Fig. 2). On the other hand, PI staining (red) detected
the late apoptotic or necrotic cells. This result demonstrates that apoptosis and necrosis areconcomitantly occurred in the case of ALA-PDC. In higher concentration of ALA, necrotic cells
were increased and apoptotic cells were decreased. In KKLS and MKN28 cells, apoptotic cells were
not observed at the IC50 condition of ALA-PDC (data not shown). These results suggest that when
the lower amounts of ALA are incubated with cancer cells with wild-type p53, the efficacy of
ALA-PDT is highly attributed to apoptosis-mediated cell death.

Figure 2. Fluorescence staining of apoptotic
and necrotic MKN45 cells using PI and
Hoechst 33258. Arrows indicate typicalapoptotic cells associated with chromatincondensation and/or cellular fragmentation.
PI derived red fluorescence is observed in
necrotic cells.
Effect of p53 inhibition to the cellular sensitivity to ALA-PDC
To investigate the potential involvement of p53 on apoptosis after ALA-PDC, we estimated the2527

effect of p53 inhibition during ALA-PDC on the cell viability. Three cell lines were used in thisexperiment: MKN45, KKLS, and MKN28. Cells were incubated with 50 μM PFT-α at increased
concentration of ALA for 4 h. Cells were then replaced with fresh medium, and exposed to LED
light for 10 min. Cell viability 24 h after ALA-PDC was measured by MTT assay. As the results,
PFT-α significantly conferred cellular resistance on MKN45 cells (Fig. 3). This result strongly
suggests that wild type p53 is critically involved in ALA-PDC induced apoptosis. In contrast, theeffects of PFT-α on both KKLS and MKN28 were minimal compared with MKN45. These results
suggest that the variant type of p53 could determine the photo-sensitivity to ALA-PDC. It is ofimportance that mutant p53 might be no efficacy of ALA-PDT.

Figure 3. Effect of pifithrin-α on cell viability of human gastric cancer cells after ALA-PDC. Cells
were incubated with ALA in presence or absence of 50 µM PFT-α. The cellular sensitivity by
ALA-PDC is measured by MTT assay as described in Experimentals section.
Ser15 phosphorylation of p53 following ALA-PDC
Human p53 possesses a series of serine / threonine phosphorylation sites. It is known the
majority of these sites are phosphorylated following cellular stress. Ser15 phosphorylation of p53 is
known to reduce p53 affinity for its primary negative regulator HDM2 and promotes therecruitment of transcriptional coactivators. In this study, we assessed the phosphorylation status andthe stabilization of p53 after ALA-PDC from immunoblotting analysis with specific antibody to p53
and Ser15-phosphorylated p53 (Fig. 4). ALA-PDC to MKN45 cells induced Ser15 phosphorylationin p53. The only LED irradiation hardly affected p53 status. Furthermore, the Ser15
phosphorylation and the deprivation of p53 were completely suppressed with a p53-specificinhibitor pifithrin-α (PFT-α). These results suggest that apoptosis following ALA-PDC in MKN45
is mediated by wild type p53. Previous studies have shown that PFT-α inhibits p53 accumulation invarious cell systems subjected to ultraviolet radiation, cisplatin, resveratrol, or doxorubicin
treatment [16; 20-23]. To our knowledge, this is the first report showing PFT-α could inhibit
ALA-PDC-induced p53 status in MKN45 cells. Reportedly it is suggest that PFT-α interferes withmultiple steps of apoptosis such as JNK and ERK [23]. Further study is ongoing in our laboratory
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for the specific mechanism that pretreatment of cells with PFT-α followed by ALA-PDC attenuatedp53-dependent apoptosis.
Figure 4. Immunoblotting of p53 protein in MKN45
cells.
The protein levels of p53 phosphorylation wereobtained by immunoblotting. We used a polyclonal
antibody specific to the phosphorylated p53 at Ser15 as
described in Experimental section.
Conclusion
We demonstrated the wild type p53 is of importance to predict the efficacy of ALA-PDT
because MKN45 cells, wild type p53, showed higher cellular sensitivity by ALA-PDC than KKLS
and MKN28 cells expressing mutant p53 (Fig. 1). Many clinical studies have revealed associations
between p53 mutation and poor clinical outcome in a variety of cancer. Over 50% of human cancers
reportedly possess p53 mutation, showing the ablation of apoptosis. Our results also suggested thatmutant p53 decreased the efficacy of ALA-PDT as well. However, ALA-PDT has a potential toinduce necrosis when higher amount of PpIX is accumulated in cancer cells (Fig.1 and 2). In thisregard, with the effort that cancer cells accumulate PpIX, ALA-PDT would be successful in the caseof p53 mutant cancer. In any of these cases, the status of p53 could be a significant factor in
determining the therapeutic strategy.
The efficacy of ALA-PDT is also considered to depend on the intracellular PpIX concentrationthat can produce cytotoxic singlet oxygen and other ROS via photoactivation reactions. In mutant
p53 cancer, cellular PpIX accumulation is required to induce necrosis. It is speculated thatenhancement of PpIX accumulation would be achieved by an inhibitor of PpIX efflux transporters.
The potential to induce both apoptosis and necrosis indicate the clinically advantageous merit of
ALA-PDT. In conclusion, we hypothesize that the variant type of p53 is potential to predict thepatient’s response to ALA-PDT.
Acknowlegement
The study in the authors’ laboratories was supported by Research Fellowships of the Japan
Society for the Promotion of Science (JSPS) for Young Scientists as well as a Grant-in-Aid forYoung Scientists (B) (No. 22700910 to S.Ogura) from the Ministry of Education, Culture, Sports,
Science and Technology. This work was partly supported by a Grant-in-Aid for Scientific Research(C) (No. 23501305 to Y.Endo) from JSPS. Y. Hagiya is a JSPS Research Fellow.
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