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https://www.abbs.info ISSN 0582-9879 |
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Short Communication |
Comparison
of 5-Aminolevulinic Acid and Its Hexylester Mediated Photodynamic Action on
Human Hepatoma Cells
REN
Qing-Guang1,2, WU Su-Min1,3, PENG Qian4, CHEN
Ji-Yao1*
(1Department
of Physics, 2Analysis and Measurement Center,
3State
Key Laboratory of Applied Surface Physics, Fudan University, Shanghai 200433,
China;
4Department
of Pathology, Institute for Cancer Research, University of Oslo, Montebello, 0310
Oslo, Norway)
Abstract 5-Aminolevulinic acid (ALA) is a precursor to
heme synthesis pathway and currently used to induce endogenous protoporphyrin
IX (PpIX, a potent photosensitizer) for photodynamic therapy of cancer. ALA
has, however, a limited ability to cross cellular membranes due to its low
lipid solubility. The use of lipophilic ALA esters may increase cellular
uptake, that results in an enhanced PpIX synthesis. In the present study, a
comparison of ALA and its hexyl ester (He-ALA) was made in the QGY human
hepatoma cell line with respect to PpIX production and its photocytotoxicity.
The fluorescence emission spectrum of the cells incubated with He-ALA was
identical to that of PpIX, indicating that He-ALA could induce PpIX in the
cells. Fluorescence images demonstrated that the He-ALA induced PpIX was
localized in the cytoplasm of the cells. Moreover, a similar amount of PpIX was
found in the cells incubated with 0.2 mmol/L He-ALA or 2 mmol/L ALA and a
similar level of cell survival was reached following light exposure. These
results suggest that He-ALA is much more efficient at producing PpIX and
photocytotoxicity than ALA itself in the cells.
Key words 5-aminolevulinic
acid (ALA); protoporphyrin; photosensitization
Photodynamic therapy
(PDT), as a new cancer treatment modality, has been employed in clinic
worldwide[1]. The concept of the treatment is that, a
photosensitizer accumulated in a tumor initiates photosensitization while being
irradiated by light with a suitable wavelength, and produces active oxygen
species to destroy the tumor[2]. During the last decade 5-aminolevulinic
acid (ALA), a precursor to heme biosynthetic pathway, was used to stimulate
endogenous protoporphyrin IX (PpIX) production in tumor[3]. Since
PpIX is an effective sensitizer, this approach was developed rapidly and has
become a new branch in PDT field, called ALA-PDT[4]. ALA-PDT has
already shown encouraging results in the treatment of several skin cancers and
other diseases[5]. However, the ability of ALA to penetrate into
cancer cells is low due to its hydrophilic property. Some esterfied ALA derivatives
have thus been developed for improving the cellular uptake, and ALA hexyl ester
(He-ALA) has been found to be more efficient than ALA itself to induce PpIX in
colon adenocarcinoma cells (WiDr) and cervix carcinoma cells (NHIK 3025)[6].
In addition, He-ALA produces less PpIX than ALA in normal skin[7],
leading to a high PpIX tumor/normal skin ratio in vivo[8]. In
the present study, a comparison of ALA and He-ALA was made in the QGY human
hepatoma cells in vitro with respect to PpIX production and photocytotoxicity.
1
Materials and Methods
1.1
Chemicals
ALA and ALA-Hexyl
ester (He-ALA), obtained from PhotoCure ASA (Oslo, Norway), were dissolved in
the PBS with pH 7.0. The stock solutions of 36 mmol/L were made and kept in 4 ℃
before use.
1.2
Cell cultivation
QGY-7903 human
hepatoma cells, obtained from Cell Bank of Chinese Academy of Sciences[9],
were maintained in RPMI 1640 medium, supplemented with 10% fetal calf serum
(FCS,Gibco BRL), penicillin 100 000 units/L, streptomycin 100 mg/L and 1% glutamine.
Cells were incubated at 37 ℃
in a humidified incubator containing 5% CO2. Cells in the
exponential growth phase were used in the experiments.
1.3
Fluorescence imaging
Cells (104)
were seeded on the glass slice, which was placed in the middle of 10 cm2
culture dishes (Nunclon). Forty-eight hours after seeding, the cells were
incubated with He-ALA (0.4 mmol/L) in serum-free medium for 6 hours. After
being washed with fresh medium, the cells on the slices were examined by an
Olympas fluorescence microscope equipped with a digital camera (Nikon). The
magnification used was 320. The filters for detection of PpIX fluorescence
consisted of a 450 nm band pass filter for excitation and a 590 nm long pass
filter for emission.
1.4
Measurements of PpIX formation in cells
Cell samples (2×105
cells) were inoculated in 10 cm2
culture dishes (Nunclon) for overnight for proper attachment to the substratum
in RPMI 1640 medium containing 10% FCS. The cells were then incubated with ALA
(2 mmol/L) or He-ALA (0.2 mmol/L) in serum-free medium for different hours.
After incubation the cells were washed with fresh medium for 3 times and
suspended in PBS (109 cells/L) for fluorescence measurements. The
fluorescence spectra and relative intensities of ALA- or He-ALA-treated cells
were measured with a luminescence spectrometer (Carry Eclipse, VARIAN). The
excitation wavelength was set at 410 nm (a main absorption peak of PpIX) and
the emission spectra were scanned
(or measured). By this way it was possible to study the kinetics of PpIX
formation in the cells[10]. Besides, that the relationship between
the relative PpIX amount in cells with different drug incubation concentration
was also studied by this way.
1.5
Photodynamic treatment and cell survival assay
The cells were added
into 96 wells flat-bottomed culture plates with 2×104
cells per well. When attached to the substratum the cells in PDT groups were
added with ALA (2 mmol/L) or He-ALA (0.2 mmol/L) in serum-free medium, and
incubated for 5 hours. The serum-free medium was also used in the cells of
control groups. The cells of both PDT and control groups were subsequently
irradiated with different light doses. The light source was a halogen lamp with
a heat-isolation filter and a 500 nm long pass filter, as described previously[11].
The fluence rate was 7 mW/cm2. After light exposure the cells had
been incubated with fresh medium containing 10% FCS for 2 days before the cell
viability was determined by MTT assay. The details of MTT assay were described
previously[11], and the optical density at 540 nm and 590 nm was
measured using iEMS Analyzer (Bio-Rad).
1.6
Statistical analysis
Data were presented as
x±s for all experiments which were repeated at least 3 times.
2
Results and Discussion
Fig.1 is the
fluorescence image of cells after He-ALA incubation, which shows that PpIX was
produced, because in cells only PpIX emit red fluorescence when excited by blue
light. The PpIX localized in cytoplasm of the cells, demonstrating that the
PpIX cellular distribution pattern induced by He-ALA was as same as that
induced by ALA[10].
Fig.1 PpIX fluorescence image of the QGY
cells
The cells were incubated with He-ALA (0.4
mmol/L) in the dark for 6 hours. The excitation was the 435 nm (band pass). The
590 nm long pass filter was used for fluorescence image detection.
Fig.2 shows the
fluorescence emission spectra of ALA treated cells and He-ALA treated cells,
with the peaks at 635 nm and 705 nm. These emission peaks (635 nm and 705 nm)
are the characteristic of PpIX in living system[12], confirming that PpIX can be
endogenously produced from the ALA
and He-ALA in QGY hepatoma cells.
Fig.2 Fluorescence emission spectra of the
QGY cell suspension
The cells were incubated with ALA (2
mmol/L) or He-ALA (0.2 mmol/L) in the dark for 5 hours. After being washed,
cells were resuspended in PBS (109 cells/L) for fluorescence
measurements. Control cells were not treated with ALA and He-ALA. Excitation:
410 nm.
By measuring the
intensities of fluorescence peak at 635 nm, the relative PpIX amounts in cells being incubated with
different ALA or He-ALA concentration were detected as shown in Fig.3. It is
shown that the PpIX cellular amount increased with the drug incubation
concentration, but satuated around 2 mmol/L ALA concentration and 0.2 mmol/L
He-ALA concentration. So, 2 mmol/L ALA concentration and 0.2 mmol/L He-ALA
concen-tration were selected for following experiments.
Fig.3 Relative PpIX cellular amount with
different drug incubation concentration
After being incubated for 5 hours with
different concentration of ALA or He-ALA respectively and washed, the fluorescence intensities of each cell
samples (109 cells/L) were measured at 635 nm. Excitation: 410
nm. Column 1, ALA (0.2 mmol/L),
He-ALA (0.02 mmol/L); Column 3, ALA (0.8 mmol/L), He-ALA (0.08 mmol/L); Column
5, ALA (2 mmol/L), He-ALA (0.2 mmol/L).
Fig.4 shows the
formation kinetics of PpIX produced in cells at different incubation times.
PpIX amount in cells increase with the ALA (2 mmol/L) or He-ALA (0.2 mmol/L)
incubation time up to 12 hours. In some cell lines, the dark toxicity of ALA
(around mmol/L incubation concentration) to cells would be heavier when
incubation time was longer than a few hours[6, 10]. Here it was
found that the resistance of QGY cells to ALA in dark was strong. After 12
hours incubation of ALA (2
mmol/L) or He-ALA (0.2 mmol/L), the death rate of cells was still less
than 5%. Such high resistance to ALA in dark was also found in some other cell
lines[13]. From Fig.4, it is shown that the kinetics of PpIX
formation for two cases of ALA incubation and He-ALA incubation are similar,
and that the much higher PpIX production efficiency of He-ALA is confirmed
since the incubation concentration of He-ALA is 10 times lower than that of
ALA.
Fig.4 PpIX formation kinetics in the QGY
cells
Cells were incubated with ALA (2 mmol/L)
or He-ALA (0.2 mmol/L) for different times. The fluorescence intensities of
cell suspension (109 cells/L) were measured at 635 nm. Excitation:
410 nm.
It is believed that,
PpIX is initially synthesized from ALA in the mitochondria of the cell, and
then diffuses into the cytoplasm of the cell[4], which was convinced
by some experimental data[9, 14]. Though for QGY hepatoma cells, the
longer ALA or He-ALA incubation time will produce more PpIX amount in cells, 5
hours incubation time was selected here to carry out the photodynamic
inactivation experiment in next step. First, it may have more PpIX confined in
mitochondria during the short time incubation. Mitochondria is a very crucial
target of photosensitization to damage the cells[1]. We also found
in previous work that mitochondria was the key organelle to initiate apoptosis
during cell photosensitization[15,16]. Second, in most studies
concerned ALA-PDT the incubation time was around 4 hours. The selection of 5
hours incubation time will make this work easier comparing with other similar
work[13, 17]. Fig.5
shows the photo-inactivation effect to cells after 5 hours ALA or He-ALA
incubation and different dosage irradiation. When irradiation dose was
relatively small, the damaged extent of cells was light, which may due to cell
repairing function. When irradiation dose increased, the cells were seriously
damaged. However, the sensitivity of QGY hepatoma cells to ALA-PDT was lower.
After 5 hours ALA (2 mmol/L) incubation and 147 kJ/m2 dosage
irradiation (35 min), the death rate of QGY cells only reached 55%. For HeLa cells, after 2 hours ALA (0.7
mmol/L) incubation and 312 kJ/m2 dosage irradiation, the 99% cells
were destroyed[14]. While in the case of leukemia cells, after 4
hours ALA (1 mmol/L) incubation and 45 kJ/m2 dosage irradiation, the
death rate was over 90%[9], showing the higher ALA-PDT sensitivity.
But meanwhile, the dark toxicity of ALA to leukemia cells was also higher. When
incubated with ALA (1 mmol/L) for more than 5 hours without irradiation, the
death rate of leukemia cells was over 10%, implying the dark toxicity may
correlate with PDT sensitivity. Considering the PDT sensitivity and the ALA
dark toxicity of QGY cells are both lower, it seems that the tolerance of this
cell line is strong.
Fig.5 Photo-inactivation of ALA or He-ALA to
the QGY cells
Cells were incubated with ALA (2 mmol/L)
or He-ALA (0.2 mmol/L) for 5 hours followed by irradiation with different light
doses. The cell survival was measured by MTT assay.
Though the QGY cells
die hard, ALA-PDT efficacy is not high. The death rate of QGY cells reached 75%
when incubated with He-ALA (0.2 mmol/L) for 5 hours and irradiated with 147
kJ/m2 dose. The PDT effect of He-ALA is more than 10 times higher
than that of ALA, exhibiting He-ALA is very effective in photo-inactivation of
the hepatoma cells. It was shown that He-ALA had much higher PDT efficiency
than ALA in some cell lines[6]. Here in this resistant QGY cell
line, He-ALA is also much powerful
than ALA in photosensitization. In conclusion, He-ALA is thus a very promising
drug, may instead of ALA, used in photodynamic therapy.
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Received: February
25,2002 Accepted:
April 28,2002
This work was
supported by a grant from the National Natural Science Foundation of China
(No.39970186)
*Corresponding
author: Tel, 86-21-65642366; Fax, 86-21-65104949; e-mail,
[email protected] Communication