Development of a novel Schiff base derivative for enhancing the anticancer potential of 5-aminolevulinic acid-based photodynamic therapy

Article abstract of DOI:10.1016/j.pdpdt.2017.10.014

5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), is now widely used for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various cancers. Recently, we found that treatment of cancer cells with the Schiff base derivative TX-816 along with ALA could significantly increase the efficacy of ALA-PDT. This enhancing effect of TX-816 on ALA-PDT is attributed to 3,5-dichlorosalicylaldehyde (DCSA), a molecule produced by the degradation of TX-816. Similar to TX-816, DCSA significantly enhances the effect of ALA-PDT. Furthermore, DCSA could restore the sensitivity of cancer cells that acquired resistance to ALA-PDT. These results indicate that DCSA, as well as TX-816, is a potent lead compound for the development of an ALA-PDT sensitizer. TX-816 might be a useful compound for designing prodrug-type ALA-PDT enhancers.

Full text of DOI:10.1016/j.pdpdt.2017.10.014

PhotodiagnosisandPhotodynamicTherapy20(2017)182–188
Contents lists available at ScienceDirect
Photodiagnosis and Photodynamic Therapy
journal homepage: www.elsevier.com/locate/pdpdt
Development of a novel Schiff base derivative for enhancing the anticancer
potential of 5-aminolevulinic acid-based photodynamic therapy
Yusei Shinoharaa, Yoshio Endob,1, Chiaki Abec, Ikkyu Shibaa, Masahiro Ishizukad, Tohru Tanakad,
Yutaka Yonemurae, Shun-Ichiro Oguraf, Masahide Tominagag, Hisatsugu Yamadah,
Yoshihiro Utoh,⁎,1
a
Graduate School of Advanced Technology and Science, Tokushima University, Tokushima, Japan
Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
Department of Molecular Life Science, Institute of Biomedical Research and Innovation, Kobe, Japan
SBI Pharmaceuticals Co., Ltd., Tokyo, Japan
NPO Organization to Support Peritoneal Dissemination Treatment, Osaka, Japan
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Institute of Health Science, Tokushima University Graduate School, Tokushima, Japan
Institute of Bioscience & Bioindustry, Tokushima University Graduate School, Tokushima, Japan
b
c
d
e
f
g
h
A R T I C L E I N F O
A B S T R A C T
Keywords:
5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), is now widely used for photodynamic
diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various cancers. Recently, we found that treat-
ment of cancer cells with the Schiff base derivative TX-816 along with ALA could significantly increase the
efficacy of ALA-PDT. This enhancing effect of TX-816 on ALA-PDT is attributed to 3,5-dichlorosalicylaldehyde
(DCSA), a molecule produced by the degradation of TX-816. Similar to TX-816, DCSA significantly enhances the
effect of ALA-PDT. Furthermore, DCSA could restore the sensitivity of cancer cells that acquired resistance to
ALA-PDT. These results indicate that DCSA, as well as TX-816, is a potent lead compound for the development of
an ALA-PDT sensitizer. TX-816 might be a useful compound for designing prodrug-type ALA-PDT enhancers.
5-Aminolevulinic acid
Protoporphyrin IX
Photodynamic therapy
Schiff base
1. Introduction
guide for tumor resection. Furthermore, absorption of light beyond
630 nm by PpIX leads to the production of cell-modifying reactive
5-Aminolevulinic acid (ALA)-based photodynamic diagnosis (ALA-
PDD) and therapy (ALA-PDT) are widely accepted novel diagnostic and
therapeutic strategies against various cancers [1]. ALA is an intermediate
precursor in the heme biosynthesis pathway. Its oral administration to
cancer patients leads to the selective accumulation of protoporphyrin IX
(PpIX) within the malignant tissue. First, ALA is incorporated into cancer
cells mainly through a proton-coupled oligopeptide cotransporter called
solute carrier family 15 member A1 (SLC15A1), which is also well-known
as PEPT1 [2]. Subsequently, it is enzymatically converted to PpIX
through the heme biosynthesis pathway. Following excitation with blue
light (405–410 nm), the accumulated PpIX emits a red fluorescence
(635 nm), via which the cancerous tissues can be effectively visualized
(ALA-PDD). Surgeons can potentially exploit ALA-PDD and use it as a
oxygen species (ROS) that could induce cell death in various cancer cells
(ALA-PDT). Thus, ALA-PDT can be applied as a successful treatment for
patients with various neoplastic and non-neoplastic diseases. In a pre-
vious study, we demonstrated that the sensitivity of cancer cells to ALA-
PDT is determined by the balance between the influx of ALA through the
transporter PEPT1 and the efflux of intracellular PpIX through the ABC
transporter ABCG2 [3,4].
Recently, we found that the Schiff base derivative N-3′,5′-dichloro-
2′-hydroxybenzylidene-2-chloro-4-nitroaniline, known as TX-816
(Fig. 1), could significantly increase the effect of ALA-PDT by accel-
erating the intracellular accumulation of PpIX. Herein, we report the
TX-816-induced enhancement of ALA-PDT cytocidal activity against
human cancer cells.
⁎
Corresponding author at: Institute of Bioscience & Bioindustry, Tokushima University Graduate School, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima, 770-8506,
Japan.
E-mail address: uto.yoshihiro@tokushima-u.ac.jp (Y. Uto).
Contributed equally.
1
http://dx.doi.org/10.1016/j.pdpdt.2017.10.014
Received 12 September 2017; Received in revised form 13 October 2017; Accepted 18 October 2017
Availableonline19October2017
1572-1000/©2017ElsevierB.V.Allrightsreserved.

Y. Shinohara et al.
PhotodiagnosisandPhotodynamicTherapy20(2017)182–188
Fig. 1. Chemical structures of TX-816 (A) and its degradation products, DCSA (B) and
CNA (C).
2. Materials and methods
2.1. Chemicals
TX-816
was
synthesized
in
our
laboratory.
3,5-
Dichlorosalicylaldehyde (DCSA) (300 mg, 1.57 mmol), 2-chloro-4-ni-
troaniline (CNA) (271 mg, 1.57 mmol), and dried 4A molecular sieves
were dissolved in dehydrated toluene and reacted with reflux. The
solvents were evaporated, and the residue was purified by silica-gel
column chromatography with dehydrated 50% hexane/ethyl acetate to
obtain TX-816 (121 mg, 22.3%) as a red solid. 1H NMR (400 MHz, di-
methyl sulfoxide (DMSO)-d6): δ13.5 (br s, 1H), 9.13 (s, 1H), 8.48 (s,
1H), 8.37 (d, 1H, J = 8.8 Hz), 7.85–7.82 (m, 3H). TX-816 produces
DCSA and CNA by hydrolytic degradation in the solvent.
Hematoporphyrin IX dihydrochloride (HEMP) and dipyridamole (DPM)
were purchased from Wako Pure Chemical Industries (Osaka, Japan),
whereas DCSA and CNA were purchased from Tokyo Chemical Industry
(Tokyo, Japan).
Fig. 2. The LED light source. The LED irradiation unit equips four module boards con-
sisting of twenty (4 × 5) LEDs.
manufacturer’s instructions. After 72 h of incubation, the medium was
replaced by fresh medium containing the WST-8 reagent. After 3 h,
absorbance in each well was measured at 450 nm (with a reference
2.2. Cells and cell culture
wavelength of 620 nm) using
a microplate spectrophotometer,
ImmunoMini NJ-2300 (BioTec, Tokyo, Japan).
The poorly differentiated gastric cancer cell line MKN-45 was kindly
provided by Dr. Suzuki (Fukushima Medical College, Fukushima,
Japan). The human gastric cancer cell line KKLS, which was established
from a metastasized lymph node of a patient with multiple metastasis in
the liver and lymph nodes, was kindly provided by Dr. Mai (Cancer
Research Institute of Kanazawa University) [5]. The poorly differ-
entiated signet ring cell carcinoma cell line NUGC-4 was obtained from
the Japanese Cancer Research Resources Bank (Tokyo, Japan).
Cells were maintained in RPMI-1640 medium supplemented with
10% (v/v) heat-inactivated fetal bovine serum and 50 μg/mL kana-
mycin at 37 °C in a humidified atmosphere containing 5% CO2.
The percentage of cell growth inhibition was calculated by applying
the following formula: cell growth inhibition (%) = (1−[T/C]) × 100,
where C and T are the mean absorbance values of the control and
treated groups, respectively. The IC50 value was determined graphically
from the dose-response curve with at least three drug concentration
points.
The isobologram method [6] was used to determine whether the
effect of TX-816 on ALA-PDT sensitivity was additive, synergistic, or
competitive. In the combination assay, serial dilutions of TX-816 (0,
6.25, 12.5, 25, 50, 100, and 200 μM) were added to the culture
medium. On the other hand, ALA was added at concentrations of 0,
6.25, 12.5, 25, 50, and 100 μM. After 4 h of incubation, the medium
was replaced by fresh complete medium, and the 96-well plate was
exposed to LED irradiation for 5 min. Cell viability was measured by a
colorimetric assay as stated above. The IC10, IC20, IC30, IC40, and IC50
values of ALA alone and TX-816 alone were determined graphically
from the dose-response curve. The IC10, IC20, IC30, IC40, and IC50 values
of ALA were defined as a1, a2, a3, a4, and a5 and those of TX-816 were
defined as b1, b2, b3, b4, and b5, respectively. At first, (0, a5) and (b5, 0)
were plotted on the Y axis and X axis of the graph, respectively. Next,
(b5-b1, a1), (b5-b2, a2), (b5-b3, a3), (b5-b4, a4), (b1, a5-a1), (b2, a5-
a2), (b3, a5-a3), and (b4, a5-a4) were plotted on the graph. Finally, the
IC50 values of the combinations of ALA and TX-816 were represented on
the graph. The inner area surrounded by the two lines of their IC10–IC50
values shows the range of predictive IC50 values whose corresponding
combinations have an additive effect; IC50 values of the combination
treatments located within the gray zone, under the gray zone, and
above the gray zone correspond to combinations having additive, sy-
nergistic, and competitive effects, respectively.
2.3. ALA-PDT sensitivity assay by LED irradiation
Cells were seeded at a density of 5 × 103 cells/well in a 96-well
plate and cultured at 37 °C in 5% CO2 for 24 h. Test compounds such as
TX-816, DCSA, CNA, and DPM were dissolved in DMSO and added to
the culture medium at the stated concentrations. The final concentra-
tion of DMSO in the culture medium was 0.5%. Thereafter, serial di-
lutions of ALA were added to the culture medium at concentrations
between 7.8 and 1000 μM. After 4 h of incubation, the medium was
replaced by fresh complete medium and the 96-well plate was exposed
to LED irradiation (630 nm, 80 mW/cm2) emitted by an LED irradiation
unit provided by SBI ALApromo (Tokyo, Japan) for 5 min (Fig. 2). The
LED light spot was an equally illuminated rectangular spot measuring
128 × 86 mm in size covering the whole area of the 96-well culture
plate.
After LED irradiation, cells were further incubated for 48–72 h, and
cell viability was measured by a colorimetric assay using Cell Counting
Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the
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Y. Shinohara et al.
PhotodiagnosisandPhotodynamicTherapy20(2017)182–188
Fig. 3. Establishment of MKN-45 cells with acquired resistance to ALA-PDT. To obtain
cell lines resistant to ALA-PDT, MKN-45 cells were treated repeatedly with ALA-PDT and
resistant cell colonies were isolated and cultured separately (R1, R2, and R13). Clone R13
was subject to re-cloning and subsequently, R13-1 and R13-2 were isolated.
Fig. 6. The enhancing effects of TX-816, DCSA, and CNA on ALA-PDT against MKN-45
cells. MKN-45 cells were seeded at a density of 5 × 103 cells/well in a 96-well plate. After
24 h of incubation, TX-816, DCSA, or CNA (20 μM) was added to the culture medium.
Thereafter, serial dilutions of ALA were added to the culture medium as described in the
“Materials and Methods” section. After 4 h of incubation, the 96-well plate was exposed
to LED irradiation for 5 min and further incubated for 72 h.
*R, originated from MKN-45R, is a cell line that was repeatedly exposed to 5-min PDT
using 200 μM ALA. **R400 is a cell line resistant to 5-min PDT using 400 μM ALA.
cells were exposed to red LED light (630 nm) for 5 min. The surviving
and re-growing cells, which were resistant to ALA, were collected and
seeded into a new dish, treated again with ALA-PDT, and re-seeded into
a new 15-cm dish after each treatment.
2.5. Measurement of intracellular PpIX
Cells (2 × 106 cells) were seeded in 6-cm dishes and incubated for
24 h. TX-816, DCSA, and DPM were added to the medium at the stated
concentrations before ALA addition. After 4 h of incubation, cells were
harvested using 2 mL of 0.25% Trypsin-1 mM EDTA. A cell suspension
was collected in a 5-mL tube and centrifuged at 800 rpm for 5 min. The
supernatant was aspirated and cells were washed with phosphate-buf-
fered saline. Finally, the cell pellet was suspended in 500 μL of 2.5%
Triton X-100 solution and the cell suspension was transferred into a 0.5-
mL micro tube, vortexed, incubated for 5 min at room temperature, and
centrifuged at 15,000 rpm for 10 min. The fluorescence intensity of
PpIX in the supernatant was measured using a SEC2000-UV/vis spec-
trophotometer (excitation: 410 nm, emission: 630 nm).
Fig. 4. The enhancing effect of TX-816 on ALA-PDT. MKN-45 cells were seeded at a
density of 5 × 103 cells/well in a 96-well plate. After 24 h of incubation, TX-816 (5, 10,
or 20 μM) was added to the culture medium. Thereafter, serial dilutions of ALA were
added to the culture medium as described in the “Materials and Methods” section. After
4 h of incubation, the 96-well plate was exposed to LED irradiation for 5 min and further
incubated for 72 h.
2.4. Establishment of ALA-PDT-resistant MKN-45 cells
Resistant cells were established by repeated treatment with ALA-
PDT and sub-cloning, as shown in Fig. 3. Briefly, MKN-45 cells were
seeded into a 35-mm dish and incubated with 200 μM ALA for 4 h. The
Fig. 5. The isobologram analysis of the
combination treatment with ALA and TX-
816. Various concentrations of TX-816 and
ALA were added to the culture medium as
described in the “Materials and methods”
section. The IC50 values of the combination
treatments were determined by the colori-
metric assay using the WST-8 reagent, and
the type of the effect was evaluated by the
isobologram analysis. The IC50 values of
ALA and TX-816 combinations are re-
presented by open triangles (△) and open
squares (□). The inner area surrounded by
the two lines shows the range of predictive
IC50 values if the combinations have an ad-
ditive effect. Since the IC50 values are lo-
cated in the low-concentration side under
the gray zone, these combinations were
judged to have synergistic effects.
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Y. Shinohara et al.
PhotodiagnosisandPhotodynamicTherapy20(2017)182–188
Fig. 7. The ALA-PDT enhancing effects of TX-806, DCSA, and DPM in parental MKN-45 cells, its ALA-PDT-resistant cell lines, KKLS cells, and NUGC cells. (A) RT-PCR analysis of PEPT1
and ABCG2 in gastric cancer cells. (B) The enhancing effects of TX-816, DCSA, and DPM on ALA-PDT in ALA-PDT-resistant cell lines. (C) The enhancing effects of DCSA and DPM on ALA-
PDT in KKLS and NUGC cells. Cells were seeded at a density of 5 × 103 cells/well in a 96-well plate. After 24 h of incubation, TX-816, DCSA (20 μM), or DPM (10 μM) was added to the
culture medium. Thereafter, serial dilutions of ALA were added to the culture medium as described in the “Materials and Methods” section. After 4 h of incubation, the 96-well plate was
exposed to LED irradiation for 5 min and further incubated for 72 h.
Ace (TOYOBO, Osaka,Japan). Next, cDNA was amplified by PCR in a
Thermal Cycler Dice (TaKaRa Bio, Otsu, Japan). The specific sense and
antisense primers were as follows: 5′-TGACGCCAATTCTCGGAGCTCTTATC-
3′ (sense) and 5′-CAGGAACATCACCCTCGTAACCATCT-3′ (anti-sense) for
PEPT1 (NM_005073, SLC15A1) (the product size: 665 bp); 5′- GGT
TACGTGGTACAAGATGATGTTGTGATG-3′ (sense) and 5′- CCAGC
Table 1
The enhancing effects of TX-816, DCSA, and DPM on ALA-PDT in ALA-PDT-resistant cell
lines.
IC50 (μM) of ALA
Combination with ALA
TCTGTTCTGGATTCCAGTAGAATCA-3′
(anti-sense)
for
ABCG2
Cell
ALA alone
TX-816
DCSA
DPM
(NM_004827) (the product size: 920 bp); and 5′-GAAAATCTGG
CACCACACCTT-3′ (sense) and 5′-TTGAAGGTAGTTTCGTGGAT-3′ (anti-
sense) for β-actin (NM_001101) (the product size: 519 bp), used as the in-
ternal control. The PCR reaction consisted of hot-start incubation at 95 °C
for 3 min and 33 cycles, each consisting of 30 s at 94 °C, 40 s at 62 °C, and
60 s at 72 °C. The resulting amplicons were separated by 1.2% agarose gel
electrophoresis and detected with ethidium bromide under ultraviolet light.
MKN-45
R
R1
R13
R400
64.3
21.9
35.9
> 1000
96.3
41.5
17.0
54.4
> 1000
102.4
46.7
77.0
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
2.6. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of
mRNA expression of PEPT1 and ABCG2
3. Results
Total RNA was extracted from cells using ISOGEN (Nippon Gene, Tokyo,
Japan) according to the manufacturer’s protocol. First-strand cDNA was
prepared from total RNA via reverse transcriptase reaction using ReverTra
3.1. The enhancing effect of TX-816 on ALA-PDT
The Schiff base derivative TX-816 could increase the effect of ALA-
185

Y. Shinohara et al.
PhotodiagnosisandPhotodynamicTherapy20(2017)182–188
Fig. 8. The effect of DCSA on HEMP-PDT in parental MKN-45 and its derived ALA-PDT-resistant cell lines. MKN-45 and its ALA-PDT-resistant cells were seeded at a density of 5 × 103
cells/well in a 96-well plate. After 24 h of incubation, DCSA (20 μM) was added to the culture medium. Then, serial dilutions of HEMP were added to the culture medium at con-
centrations of 1.25–40 μM. After 4 h of incubation, the 96-well plate was exposed to LED irradiation for 5 min and further incubated for 72 h. Cell viability was measured by a
colorimetric assay using Cell Counting Kit-8 as described in the “Materials and Methods” section.
Fig. 9. Decreased intracellular accumulation of PpIX in
highly ALA-PDT-resistant cells.
PDT against MKN-45 cells in a dose-dependent manner (Fig. 4). The
isobologram analysis revealed that the combination of TX-816 and ALA-
PDT had a synergistic effect on MKN-45 cells (Fig. 5).
3.3. The ALA-PDT-enhancing effects of TX-816, DCSA, and DPM in
parental MKN-45 cells and its ALA-PDT resistant cell lines
All cells originating from MKN-45 cells showed high resistance to
ALA-PDT, even at ALA concentrations of 1 mM. Of the four resistant cell
lines, R1 showed a considerably low mRNA expression of PEPT1 and
high expression of ABCG2. However, the other resistant cell lines (R,
R13, and R400), similar to the parental MKN-45 cells, showed high
mRNA expression of PEPT1 and no expression of ABCG2 (Fig. 7A).
Although DCSA was able to recover the sensitivity of the resistant cell
lines R, R13, and R400 to ALA-PDT, R1 cells remained highly resistant
to ALA-PDT even after the combination treatment with ALA and DCSA
(Fig. 7B). The enhancing effect of DCSA on ALA-PDT was equivalent to
that of TX-816 (Table 1). DPM, a platelet inhibitor and coronary va-
sodilator used to prevent arterial thromboembolism, inhibited ABCG2
3.2. The enhancing effect of TX-816 degradation products on ALA-PDT
We found that TX-816 undergoes hydrolytic degradation in the
solvent producing DCSA and CNA (Fig. 1). Therefore, we examined the
enhancing ability of DCSA and CNA on ALA-PDT. Only DCSA was found
to significantly increase the effect of ALA-PDT in human gastric cancer
MKN-45 cells. The ability of DCSA to enhance the efficacy of ALA-PDT
was almost equivalent to that of TX-816. On the other hand, CNA
showed a weak enhancing effect on ALA-PDT (Fig. 6).
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Fig. 10. Increased intracellular accumulation of PpIX in MKN-45 and R13 cells after combination treatment with ALA and DCSA.
Table 2
Intracellular accumulation of PpIX in MKN-45 and R13 cells after combination treatment with ALA and DCSA.
MKN-45
R13
PpIX (pmol/0.5 mL/10^6 cells)
PpIX (pmol/0.5 mL/106 cells)
ALA alone
ALA + 20 μM DCSA
T/C ratio
ALA alone
ALA + 20 μM DCSA
T/C ratio
50 μM ALA
100 μM ALA
200 μM ALA
None
5.0
18.0
NT
21.9
37.2
NT
4.4
2.1
NTa
1.8
3.9
0.4
NT
8.9
12.9
NT
4.8
3.3
0.3
NT
a
NT: not tested.
and equilibrative nucleoside transporters such as SLC29A1 and
SLC29A2. Although DPM can enhance ALA-PDT sensitivity in cancer
cells expressing both PEPT1 and ABCG2, it had no effect on ALA-PDT
sensitivity in all the resistant cells derived from MKN-45 cells. Human
gastric cancer KKLS cells express relatively low mRNA levels of PEPT-1
and ABCG-2 (Fig. 7A). In KKLS cells, only DCSA was able to enhance the
ALA-PDT effect, and only at high concentrations of ALA (Fig. 7C). The
IC50 values in KKLS cells were 505.1 μM for ALA alone, 336.9 μM for
the ALA and DCSA combination, and 829.4 μM for the ALA and DPM
combination. However, in NUGC-4 cells, which express both PEPT-1
and ABCG-2, both DCSA and DPM showed ALA-PDT-enhancing activ-
ities. The IC50 values in NUGC-4 cells were 3244 μM for ALA alone,
125.7 μM for ALA plus DCSA, and 171.4 μM for ALA plus DPM.
On the other hand, DCSA had no enhancing effect on PDT using
HEMP (HEMP-PDT) in both parental MKN-45 and its derived ALA-PDT
resistant cell lines. All the tested cell lines were highly sensitive to
HEMP-PDT. The IC50 values in parental MKN-45 cells were as follows:
5.7 μM for HEMP alone and 5.3 μM for HEMP and DCSA combination;
in R cells: 7.5 μM for HEMP alone and 8.2 μM for HEMP and DCSA
combination; in R13 cells: 7.5 μM for HEMP alone and 7.5 μM for
HEMP and DCSA combination; in R1 cells: 7.6 μM for HEMP alone and
8.1 μM for HEMP and DCSA combination (Fig. 8). These findings in-
dicate that DCSA does not stimulate the cytocidal activity of PpIX
mediated by the production of ROS.
was considerably reduced in all the ALA-PDT resistant cells (Fig. 9);
intracellular PpIX level in R13-resistant cells was only 10% of that in
parental MKN-45 cells. However, DCSA effectively increased the in-
tracellular accumulation of PpIX in both parental and derived resistant
cells (Fig. 10). When cells were treated with 100 μM of ALA, in-
tracellular accumulation of PpIX was increased 2.1 times in parental
MKN-45 cells and 4.8 times in R13 resistant cells (Table 2).
4. Discussion
In this study, we found that the Schiff base derivative TX-816 could
significantly enhance the effect of ALA-PDT by increasing the in-
tracellular accumulation of PpIX. Furthermore, the enhancing effect of
TX-816 on ALA-PDT was attributed to DCSA, a TX-816 degradation
product. The whole ALA-PDT-enhancing mechanism of TX-816 and
DCSA is still not clear. However, DCSA had no enhancing effect on
HEMP-PDT. Both parental MKN-45 cell line and its derived ALA-PDT
resistant cells were highly sensitive to HEMP-PDT. These findings in-
dicate that DCSA does not directly stimulate the cytocidal activity of
PpIX mediated by the production of ROS. R1 cells demonstrated an
extremely low mRNA expression of PEPT1. Therefore, they could not
uptake enough ALA to display the cytocidal activity and, as a result,
were insensitive to DCSA. However, R, R13, and R400 cells showed
higher mRNA expression of PEPT1. In spite of their low expression of
ABCG2, intracellular accumulation of PpIX in these resistant cells was
considerably reduced after ALA treatment. It has been demonstrated
that ABCB6 located on the mitochondrial membrane acts as a copro-
porphyrin III (CPIII)-specific transporter [7]. It is also located on plasma
membranes [8]. We previously reported that plasma membrane ABCB6
3.4. Increased intracellular accumulation of PpIX after the combination
treatment with ALA and DCSA
Intracellular accumulation of PpIX after treatment with ALA for 4 h
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exports CPIII and is upregulated in hypoxia [9]. Since hypoxia sig-
nificantly reduces ALA-induced PpIX accumulation in cancer cells
[10,11], the functional expression of ABCB6 in plasma membrane in
cases of hypoxia is an important determinant for PpIX accumulation
after ALA administration. To elucidate the molecular mechanisms un-
derlying ALA-PDT resistance, we examined the gene expression profiles
in resistant cell lines by microarray analysis. Our results demonstrated
wide variation in the gene expression profiles of the heme catabolic
pathway and iron redox system, as well as ALA influx and PpIX efflux
transporters, in the resistant cells (unpublished data). R1 cells showed
high mRNA expression of ABCB6, which may be one of the target
molecules that confer ALA-PDT resistance to the cells. However, there
was no difference between the mRNA expression of ABCB6 in R and
R13 cells and in parental MKN-45 cells. R and R13 cells highly express
ABC transporters such as ABCA3, ABCB4, ABCG4, ABCC2, and ABCC3.
ABCC2 and ABCC3 function in biliary transport of bilirubin glucur-
onides and are involved in multidrug resistance such as resistance to
vinblastine, methotrexate, and VP16 [12–14]. All the resistant cell lines
including R1 were found to express elevated levels of ferrochelatase and
heme oxygenase. Activation of the heme catabolic pathway may be an
important mechanism underlying the acquired resistance. However,
further studies are required to elucidate the relationship between the
ALA-PDT-enhancing activity of DCSA and the function of these ABC
transporters. ABCG2 does not appear to be a molecular target of DCSA,
as MKN-45 cells, which show a considerably low mRNA expression of
ABCG2 and are insensitive to the ABCG2-inhibitory drug DPM, are still
highly responsive to the ALA-PDT-enhancing effect of DCSA.
Although TX-816 was unstable in the solvent, we recently found
that newly synthesized TX-816 derivatives in which 2-chloro-4-ni-
troaniline was substituted with 4-alkyl aniline were significantly more
stable than TX-816 (unpublished data). DCSA with an aldehyde group
seems to exhibit low stability in blood due to reactivity with amines of
serum proteins. These chemically stable derivatives may be in-
corporated in the form of a Schiff base into cancer cells, and thereafter
converted into DCSA. In conclusion, we propose that DCSA is a potent
lead compound for the development of an ALA-PDT sensitizer, and TX-
816 may be a useful compound to design prodrug-type ALA-PDT en-
hancers.
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This study was partly funded by SBI Pharmaceuticals Co., Ltd.
Acknowledgment
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This study was partly supported by JSPS KAKENHI Grant Numbers
JP26430160 and JP23501305 (Yoshio Endo).
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