Comparison of the influence of photodynamic reaction on the Me45 and MEWO cell lines in vitro

Introduction

Malignant melanoma (lat. melanoma malignum) is a tumor derived from melanocytes – skin pigment cells responsible for melanin production [1, 2]. It is the most severe skin neoplasm as it may grow rapidly and metastasize through blood and lymphatic vessels [3–5]. Previous attempts of multi-agent chemotherapy for treatment of such malignant changes provided an objective response rate of 20% [6]. In addition, chemotherapeutic agents often lead to secondary tumor resistance [7]. Current melanoma treatment is based mainly on surgical removal by a large marginal of safety (5 mm to 2 cm). The most common chemotherapeutic agents used in melanoma treatment are melphalan and dacarbazine [6, 8]. There is no fully effective treatment and the application of photodynamic therapy (PDT) opens up new perspectives in the therapy of this type of cancer. Photodynamic therapy is based on cooperation of three factors: photosensitizer, which accumulates only in the tumor; light of the appropriate wavelength; and oxygen dissolved in the tissue. Photosensitizer is activated after exposure to appropriate light wavelength. The excitation energy is transferred from the absorption site and then for the production of molecular oxygen [8, 9]. The photochemical interactions of the photosensitizer, light and molecular oxygen produce singlet oxygen and other forms of reactive oxygen species (ROS). Photodynamic therapy induces disintegration

of cellular structures and modulation of genetic information. These changes are caused by oxidative stress and cytotoxic effects in the cell. In recent studies of Kästle et al. the authors observed a high level of ROS after 5-ALA-PDT in WM451LU melanoma cell line [10]. It was presented that WM451LU cells are more susceptible to PDT than normal human keratinocyte cells. The authors suggest that it could be induced by altered metabolism of heme in cancer cells. Leibovici et al. have shown that in cancer cells activity of porphobilinogen deaminase increased significantly as compared to normal cells [11]. Furthermore, Dailey and Smith showed a significant decrease of ferrochelatase activity in

various cancer cells [12]. These metabolic changes cause the accumulation

of protoporphyrin photosensitizers in cancer cells. Therefore, the concentration of accumulated photosensitizer is significantly higher than in normal cells. Today, the “gold standard” is surgical removal of melanoma. However, there are cases, especially in the elderly, in which it is not possible to perform the operation. Moreover, some melanomas are completely inoperable [13]. Especially the surgical treatment of lentiginous melanoma in the elderly is often problematic [14]. For this reason PDT may be an alternative method of cancer treatment.

The aim of this research was to compare the influence of photodynamic action with Photofrin® on survival of melanoma cells derived from primary and secondary cell lines in vitro. Photosensitizer distribution was also evaluated in both types of cells.

Material and methods

Cell lines



In the research a secondary melanoma line called Me45 was used. The cell line was obtained from the Oncology Centre Gliwice, where the line was derived from a 35-year-old woman’s lymph node cells. The primary line used in experiments was MEWO cell line, purchased from CLS (Cell Lines Service, Germany). The cells were cultured in culture flasks (25 cm³, Falcon) in DMEM (Sigma) with 2 mmol/l glutamine and 10% fetal bovine serum (FBS, Bio Whittaker, Fetal Bovine Serum, South American origin). The cells were incubated at a temperature of 37°C and in the presence of 5% CO2. Cells intended for experiments were trypsinized (Trypsin-EDTA solution, T4049, Sigma-Aldrich) and then rinsed with PBS.



Photodynamic therapy



The photosensitizer used in the therapy was Photofrin® (Ph, QLT Phototherapeutics, Inc. Vancouver, Canada). Cells were incubated for 18 h in darkness with 20 g/ml Ph in DMEM. Next for 10 minutes it was irradiated with light with power of 10 mW/cm² using a lamp (OPTEL, Opole, Poland) with a red filter (632.8 nm) [15, 16]. Cells were again incubated in the same conditions for 3, 6 or 24 h.



Proliferative test



Cell survival was assessed by checking cellular mitochondrial activity. Metabolic activity was evaluated using the tetrazolium salt reduction test in cell mitochondria (MTT Assay, Sigma Chemical Co.; 71K8409, In Vitro Toxicology Assay). Mitochondrial activity of living cells was examined in 96-well plates. 3 × 104 cells were placed in each well. The measurements were made on a Multiskan MS microplate reader (Labsystem) at wavelength 570 nm. Results are shown as percentage of control.



Localization of photosensitizer and mitochondria



Cells were incubated for 4 hours on microscope coverslips in the presence of the photosensitizer. Cells were fixed with 4% paraformaldehyde, and then rinsed with PBS. To stain the mitochondria, cells were incubated with 100 nmol/l Mito-Tracker Green (Molecular Probes, Eugene, OR, a fluorescent dye which stains mitochondria green) for 10 minutes. Such prepared preparations were evaluated using a confocal microscope (LSM510 Meta, Zeiss). For intracellular distribution of photosensitizer a filter with an excitation wavelength  =

= 405–753 nm was applied. To illustrate the cellular mitochondria, a filter with an excitation wavelength  = 488 nm was used.

Results

Proliferative test



There were observed clear differences between the survival of irradiated samples with Photofrin®, and the survival of non-irradiated samples (Fig. 1). The viability of MEWO cell line after PDT and 24 h incubation reached 22% and for Me45 cell line only 19%. After the same time of incubation, but without irradiation, 83% of MEWO and 65% of Me45 cells survived. The experiment showed that the applied photodynamic method is especially cytotoxic to the primary cell line (Me45). For each time of incubation differences in viability of photodynamically treated cells and non-irradiated samples were significant.



Localization of photosensitizer and mitochondria



In MEWO cell line the photosensitizer evenly within the cellular cytoplasm and mitochondria, whereas in Me45 cell line it accumulated primarily in mitochondrial membranes (Figs. 2, 3).

Discussion

Results presented in this paper revealed that applied PDT is cytotoxic to tested human melanoma cells. Cell survival decreased with incubation time after irradiation for both treated cell lines. Particularly sensitive to the applied therapy were primary melanoma cells (MEWO). In both cell lines the localization of Photofrin was observed mainly in mitochondrial membrane, which may lead to induction of intracellular disorders, release of apoptogenic proteins and finally to cell apoptosis [17]. Other researchers have also shown that the application of PDT in melanoma treatment is effective. The authors observed DNA damage in G361 cell line after PDT with porphyrin derivatives, which provoked apoptotic death of malignant melanoma cells [18]. Nowak-Śliwińska et al. proved that Verteporfin and Photofrin used in PDT are highly effective in mouse melanoma S91/I3 Cloudman cells [19]. The researchers observed increasing levels of singlet oxygen in cells, accompanied by a significant decrease in cell survival. Chen et al. found that PDT with methylene blue (MB) caused oxidative stress, which plays an important role in initiating cell death [20]. Our studies show that PDT with Photofrin® is a promising technique that can be combined with chemotherapy or radiotherapy, especially in the early stage of melanoma. It can also be a perfect adjuvant therapy after resection of malignant lesions [21]. In conclusion, PDT opens new non-invasive possibilities of melanoma treatment with application of modern developments in molecular biology, chemical synthesis of compounds and optical physics.

Acknowledgments

The experiments were funded by a PhD grant from the Ministry of Science and Higher Education, no. 5409 /B/P01/2011/40 (project leader: PhD. Jolanta Saczko), and funds of a research project for young scientists of the Medical University of Wroclaw: grant for young scientists no. PBmn1 (project leader: PhD. Jolanta Saczko).

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Address for correspondence



Anna Choromańska

Department of Medical Biochemistry

Wroclaw Medical University

Chałubińskiego 10

50-368 Wroclaw, Poland

tel. +48 71 784 13 87

e-mail: awawrzuta@gmail.com



Submitted: 10.02.2011

Accepted: 7.02.2012