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Short Communication |
Photodynamic
Effects of 5-Aminolevulinic Acid and Its Hexylester on Several Cell Lines
WU Shu-Min1,2, REN
Qing-Guang1,3, ZHOU Mi-Ou4, WEI Yi5, CHEN
Ji-Yao1*
(1Department
of Physics, 2State Key Laboratory of Applied Surface Physics, 3Analysis
and Measurement Center, 4Department of Physiology and Biophysics,
5Institute of Genetics, Fudan University, Shanghai 200433, China )
Abstract 5-aminolevulinic
acid (ALA) and its hexyl-ester (He-ALA) has shown promising results in
photodynamic detection and therapy of tumors. In this work, the photodynamic
effects of ALA and He-ALA on neuroblastoma cells, hepatoma cells and fibroblast
cells were comparatively studied. With the detection of fluorescence emission
spectra, protoporphyrin IX (PpIX) induced by ALA or He-ALA was observed in
these three cell lines. Confocal laser scanning microscope showed the diffuse
PpIX fluorescence in cytoplasm of neuroblastoma cells. The kinetics of PpIX
accumulation were different in these three kinds of cells. The PpIX content in
hepatoma cells and fibroblast cells continuously increased with the incubation
time of drugs until 12 h, while in neuroblastoma cells the PpIX content
saturated around 8 h after incubation with ALA or He-ALA. In addition, the PpIX
concentration in neuroblastoma cells was obviously higher than that in hepatoma
cells and fibroblast cells, indicating that the PpIX production is cell line
dependent. When incubated with ALA and irradiated with light, near 90%
neuroblastoma cells were destroyed, while for hepatoma cells and fibroblast
cells the death rate was around 50%. The results demonstrate that neuroblastoma
cells are more sensitive to ALA-PDT and the neuro-tumor cells may be well
suited for the treatment of ALA mediated photosensitization. Comparing to ALA,
He-ALA can reach the similar results concerned PpIX production and PDT damaging
in all three kinds of cells but with 10 times lower incubation concentration,
demonstrating that He-ALA has higher efficiency than ALA on inactivation of
cancer cells in vitro.
Key words
5-aminolevulinic
acid; neuroblastoma cells; hepatoma cells; fibroblast cells; photodynamic
therapy
Photodynamic
therapy (PDT) has developed to be a new clinical treatment modality for
cancer[1]. The treatment is based on preferential accumulation of the
photosensitizer in tumor. The tumor-bound photosensitizer produces active
species when irradiated with light, and consequently destroys the tumor. However,
exogenous photosensitizer used for PDT will cause prolonged skin
phototoxicity[2], and the selectivity for tumor is not so ideal. During the
last decade, considerable interest was focused on developing a new way of PDT,
which rely on an endogenously synthesized sensitizer[3]. 5-aminolevulinic acid
(ALA), a precursor to porphyrin in heme synthesis, has been used to stimulate
endogenous protoporphyrin IX (PpIX) production in tumor. Because the PpIX is a
potent photosensitizer and can emit fluorescence when excited, ALA has been
introduced for photodynamic detection and therapy of cancer[4－7]. ALA based PDT has formed a new
branch, called ALA-PDT[2]. Because of the significant difference in the
activities of key enzymes in the heme pathway between tumor and normal tissue,
the PpIX accumulation induced by ALA in tumor cells is higher than that in
normal cells[3]. ALA-PDT therefore has good tumor selectivity, and also reduces
skin photosensitivity (1 or 2 days compared to 1 or 2 months with other
photosensitizers). However, ALA is hydrophilic and does not easily penetrate
through intact skin[8] or through cell membranes[9], thus the efficiency of
PpIX production is not high. In order to overcome this problem, a number of ALA
esters with more lipophilic property have been tested, and ALA hexylester
(He-ALA) was found to be more efficient than ALA itself to induce PpIX in some
cell lines in vitro[9] and in tumor in vivo[10]. Recently, a
number of studies examining the utility of ALA in various brain tumor models have
been performed[11], and ALA exhibited the encouraging results on
fluorescence-guided resection of glioblastoma multiforme in 52 consecutive
patients[12]. The neuro-tumors may become the important area of ALA-PDT
application.
The aims of the
present study were to: (1) compare the PDT effects of ALA with He-ALA in
neuroblastoma cells, hepatoma cells and fibroblast cells to find that what kind
cancer cells are more suitable for ALA photosensitization; (2) check the
enhancing effect of He-ALA PDT on these three cell lines.
1 Materials and Methods
1.1
Drugs
5-aminolevulinic
acid (ALA) and 5-aminolevulinic acid hexyl ester (He-ALA) were obtained from
PhotoCure ASA (Oslo, Norway). Stock solutions were prepared in Dulbecco’s PBS
(Gibco BRL, Life Technologies) at a concentration of 36 mmol/L for ALA and 3.6
mmol/L for He-ALA, and stored at 4 ℃ for less than one week.
1.2 Cell lines
SK-N-SH human
neuroblastoma cells (SK), QGY-7903 human hepatoma cells (QGY) and A9 fibroblast
cells (A9), obtained from Cell Bank of the Chinese Academy of Science[13], were
used in the study. Cells were routinely cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS; Gibco BRL), 50 u/ml penicillin,
50 mg/L streptomycin and 100 mg/L neomycin at 37 ℃ in a humidified 5% CO2
incubator. Cells in the exponential growth phase were used in the experiments.
1.2 Measurement of PpIX formation in cells
ALA and He-ALA
do not emit fluorescence. Cellular PpIX induced by ALA or He-ALA emits red fluorescence
with peak around 635 nm[14]. Thus, cellular PpIX can be measured using
fluorescence method. The three type cells in culture dishes (Corning), when
already adhered on the substratum, were added to serum-free medium with ALA or
He-ALA, respectively, and then incubated at 37 ℃ in a humidified 5% CO2
incubator for designed time. After incubation, the cells were washed twice with
PBS buffer and trypsinized with 1 ml 0.25% trypsin-EDTA. Three to five minutes
later the cells were washed twice with PBS and re-suspended in PBS buffer.
These cell suspensions were adjusted to cell density of 109 cells/L
for fluorescence measurements. All procedures were performed in the darkness.
The fluorescence spectra and relative intensities of ALA or He-ALA treated cells
were measured with a luminescence spectrometer (F-2500, Hitachi). The exciting
wavelength was set at 405 nm (a main absorption peak of PpIX) and the emission
spectra were then scanned and recorded. By this way it was possible to study
the relationship between the relative PpIX amounts (relative fluorescence
intensities) in cells with different drug incubation times in different
cells[15]. In addition, the relationship between the relative PpIX content in
cells with different incubation concentration of drugs could also be studied.
1.4 Intracellular localization of PpIX
in cells
After cells were
incubated with ALA (2 mmol/L) or He-ALA (0.2 mmol/L) for 5 h and washed, the
fluorescence imaging of cell samples were measured with confocal laser scanning
microscope (Leicar, TCS NT). The excitation was 488 nm laser beam from attached
argon-krypton laser. The 590 nm long-pass filter was used to capture the
fluorescence images. The magnification used in microscopy (Leicar, DMIRB) was
630 times. This machine provides a good resolution on the Z-axis at about 0.2
μm. The PpIX intracellular distribution can be visualized from the fluorescence
images obtained.
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. After 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 h. 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 in previous
work[16]. The fluence rate was 15 mW/cm2. After light exposure the cells had
been incubated with fresh medium containing 10% FCS for 2 d before the cell
viability was determined by MTT assay, which measures the mitochondrial
dehydrogenase activity of surviving cells[17,18]. The details of MTT assay were
described previously[16], and the optical density at 540 nm and 690 nm was
measured using iEMS Analyzer (Bio-Rad).
1.6 Data
analysis and statistics
Each individual
experiment was carried out at least in triplicates. Primary data are presented
as x±s.
2 Results and Discussion
The fluorescence
emission spectra in SK cells, QGY cells and A9 cells treated with ALA (2
mmol/L) or He-ALA (0.2 mmol/L) are shown in Fig.1. The fluorescence emission
peak of 635 nm, which is the characteristic of PpIX in living system[14],
indicates that PpIX is formed in all three kinds of cells after ALA or He-ALA
incubation. At the same drug’s incubation conditions, the cellular fluorescence
intensity in SK cells is obviously stronger than that in QGY cells and A9
cells, reflecting that the intracellular PpIX content is higher in SK-N-SH
cells than in other two cells. It is also shown here that He-ALA is very
effective on stimulating the PpIX production in cells. Compared to ALA, only a
10 times lower concentration of He-ALA was needed to produce a similar PpIX
content in all three types of cells studied.
Fig.1
Fluorescence emission spectra of SK cells, QGY cells and A9 cells
The cells were incubated with 2 mmol/L ALA (solid curves) or 0.2 mmol/L
He-ALA (dashed curves) for 5 h, and then washed and resuspended in PBS with the
density of 109 cells/L. Excitation, 405 nm. SK cells, upper curves;
QGY cells, middle curves; A9 cells, bottom curves.
With the characteristic
of the highest resolution in vertical axis, confocal laser scanning microscope
was selected to measure the PpIX intracellular localization. The fluorescence
imaging of SK cells incubated with ALA or He-ALA are shown in Fig.2. The PpIX
fluorescence is observed in the cytoplasm with diffuse pattern, but not in
nuclear region for both ALA and He-ALA treated cells. This may be due to the
fact that PpIX was produced initially in the mitochondria and then diffused to
the cytosol[2]. Similar findings have been also obtained in the JCS leukemia
cells[15], WiDr adenocarcinoma cells[9]. The facts of similar
localizationpattern and similar fluorescence intensities in the images of both
ALA-incubated and He-ALA incubated cells but with 10 times lower concentration
for He-ALA, confirm the function of He-ALA is to enhance the penetration of
drug into cells.
Fig.2 Fluorescence images of SK cells detected with cofocal laser scanning
microscope
SK cells were incubated with
2 mmol/L ALA (A) and 0.2 mmol/L He-ALA (B) for 5 h. The excitation, 488 nm
laser beam. The 590 nm long pass filter was used to obtain the fluorescence
images.
The concentration effects of ALA and He-ALA
on PpIX formation were studied by measuring the fluorescence intensities (635
nm) in each cell sample, which being incubated with different ALA or He-ALA
concentration. The results are shown in Fig.3. It’s demonstrated that the
cellular PpIX production increased with ALA and He-ALA incubation concentration
at region of relative lower concentration and then saturated at higher
concentration in both SK cells and QGY cells, which consist with the common
rule as reported[2]. Since the PpIX is the product in biosynthetic pathway of
heme and the ability of heme biosynthesis is limited, the saturation of
cellular PpIX production at high ALA incubation concentration is reasonable.
He-ALA is more lipophilic and easier penetrate into cells than ALA. With 10
times lower incubation concentration than that of ALA, He-ALA stimulated
similar PpIX production in both SK and QGY cells. The saturation effect is also
obvious. At 0.2 mmol/L He-ALA concentration, the cellular PpIX accumulation
almost saturated for both SK and QGY cells. While for ALA, the saturation
happened around concentration of 2 mmol/L. Thus the concentration of 0.2 mmol/L
for He-ALA and 2 mmol/L for ALA were selected for following experiments.
Fig.3 Drug’s concentration-dependence of cellular PpIX fluorescence
intensities in SK cells and QGY cells
(A) ALA incubation. (B)
He-ALA incubation. Excitation, 405 nm. Emission, 635 nm. The density of cell
suspension: 109 cells/L.
The kinetics of PpIX
accumulation in SK, QGY and A9 cells were studied at different ALA or He-ALA
incubation time. The incubation concentration was kept at 2 mmol/L for ALA and
0.2 mmol/L for He-ALA. It’s shown from Fig.4 that for QGY and A9 cells the
cellular PpIX content increased with drug incubation time until 12 h studied,
while for SK cells around 8 h the saturation already appeared. It was reported
that for U-105MG glioblastoma cells the PpIX accumulation saturated after 24 h
ALA incubation[19], while for tumor epithelial cells the PpIX saturation
occurred around 44 h incubation[20]. However, in ALA treated C6 glioma cells,
the PpIX saturation happened as early as 6 h[21]. Combined published data and
data in Fig.4, it can be concluded that the kinetics of PpIX accumulation is
cell line dependent. In addition, it’s demonstrated from Fig.4 that the PpIX
production is also cell line dependent. At each incubation hours the PpIX
content in SK cells is obviously higher than that in QGY and A9 cells. The
activity of enzyme (ferrochelatase) is involved in PpIX accumulation
directly[3]. When this enzyme’s activity is lower, the PpIX accumulation is
relatively higher. Though the activity of ferrochelatase in cancer cells is
generally lower than that in normal cells, it’s still different for different
kinds of cancer cells. Thus the kind ofcancer cells, which has very low
activity of ferrochelatase, will accumulate more PpIX when be incubated with
ALA or He-ALA, and will be fitted better for ALA-PDT. Here among these three
kinds of cells, SK neuroblastoma cells seem the good candidate for ALA-PDT.
Fig.4
The kinetics of PpIX accumulation in SK, QGY and A9 cells
Cells were incubated with 2 mmol/L ALA
(solid lines) or 0.2 mmol/L He-ALA (dashed lines) for different time. The
fluorescence intensities of each cell suspension (109 cells/L) were
measured at 635 nm. Excitation, 405 nm.
In Fig.4, it’s
also shown that 0.2 mmol/L He-ALA can resulted in the comparable PpIX production
as 2 mmol/L ALA did. To verify this enhancing effect of He-ALA, the same
incubation concentration (0.2 mmol/L) of ALA and He-ALA was used in SK cells at
different incubation time. The results (Fig.5) demonstrated that He-ALA induced
much more PpIX content than ALA did at each incubation time tested, confirming
He-ALA is superior to ALA on PpIX production in vitro.
Fig.5
The comparison of ALA and He-ALA on PpIX production in SK cells
The incubation concentration
for ALA and He-ALA is same (0.2 mmol/L). The fluorescence intensities of each
cell suspension (109 cells/L) were measured at 635 nm. Excitation,
405 nm.
In PDT field,
it’s believed that mitochondrion is one of the most important PDT target among
subcellular organelles[1]. PDT conducted mitochondria damaging would induce
apoptosis, thus effectively destroyed cancer cells[22]. We have found
previously in leukemia cells that when photosensitizer bound on mitochondria
the apoptotic course was initiated following light irradiation[23,24]. The
mitochondrion binding is one of the crucial factors in consideration of PDT.
For ALA-PDT, PpIX is just induced in mitochondria and localize in mitochondria
first and then diffuse into cytosol, which was proved by our early work[15].
Though the cellular PpIX content will be higher at longer ALA incubation time,
it may have more PpIX amount confined in mitochondria during short drug’s
incubation time. So, 5 h incubation time of ALA and He-ALA was selected to
carry out the PDT inactivation experiments and compare the ALA-PDT efficiency
in SK, QGY and A9 cells. The most studies concerned ALA-PDT were performed
around the time of 5 h incubation[25,26]. The similar condition will make it
easy to compare our results with the parallel studies.
Fig.6 shows the
PDT-inactivation effect on SK, QGY and A9 cells after 5 h ALA (2 mmol/L) or
He-ALA (0.2 mmol/L) incubation and different dosage irradiation. Under this
drug’s incubation concentration, the dark toxicity (no light irradiation) is
very lower, less than 8% for all three kinds of cells. For each kind of cells,
the PDT damaging is proportional to the light dose. However, the PDT
sensitivity to different cells is quite different. At the same conditions of
treatment, the damaging of SK cells is more serious than that of QGY and A9
cells. The results of Fig.6 are well correlated with that of Fig.4. The PpIX is
the endogenous photosensitizer in ALA-PDT, the cellular PpIX content is higher
the damaging extent of cells should be higher accordingly when irradiated. When
being incubated with ALA for 5 h and irradiated for 35 min, the death rates are
87%, 53% and 48% for SK, QGY and A9 cells respectively. It has been reported
that the sensitivity of C6 glioma cells to ALA-PDT is also high[21], and the
conditions used for C6 cells in that experiment are comparable to here in SK
cells, reflecting neuro-tumor cells may be a kind of cells well suited for
ALA-PDT. The prognosis of malignant brain tumors with conventional treatments
remains poor. ALA-PDT has shown to be promising on treatment of glioblastoma
multiforme in clinical[12]. It seems that the potential of ALA-PDT to other
neuro-tumor is worth exploring. Comparing to ALA, He-ALA achieved similar level
inactivation to all cells but with 10 times lower incubation concentration,
convinced He-ALA has much higher PDT efficiency than ALA has. The noticeable
point is that the He-ALA enhanced the PDT effect on different cell lines no
matter neuroblastoma cells and hepatoma cells or fibroblast cells. The
enhancing effect of He-ALA on PDT might be the common rule for different cancer
cells. Thus the He-ALA is the hopeful candidate, instead of ALA, in further
study and application on PDT field.
Fig.
6 PDT damaging to SK, QGY and A9 cells
Cells were incubated with 2 mmol/L ALA
(solid lines) or 0.2 mmol/L He-ALA (dashed lines) for 5 h and then irradiated
with different light doses. The cell survival was measured 2 d later by MTT
assay (see “Materials and Methods”).
We found in
first time that SK neuroblastoma cells is a kind of cancer cells well suited
for ALA-PDT inactivation, comparing to QGY hepatoma cells and A9 fibroblast
cells. He-ALA can obtain the similar PDT result with 10 times lower dose of
ALA, showing the promising in improving the efficiency of PDT.
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___________________________________________
Received: January 24, 2003 Accepted: April 18, 2003
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]