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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(5): 650-654                                     CN 31-1300/Q

 

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