Molecular characterization of photosensitizer ...

Article

Molecular characterization of photosensitizer-mediated photodynamic therapy by gene expression profiling

Human and Experimental Toxicology 2014, Vol. 33(6) 629–637 ª The Author(s) 2014 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327113485257 het.sagepub.com

K-H Liu1, C-P Wang2, M-F Chang3, Y-W Chung3, P-J Lou2 and J-H Lin4

Abstract Photodynamic therapy (PDT) is a novel cancer treatment based on the tumor-specific accumulation of a photosensitizer followed by irradiation with visible light, which induces selective tumor cell death via production of reactive oxygen species. To elucidate the underlying mechanisms, microarray analysis was used to analyze the changes in gene expression patterns during PDT induced by various photosensitizers. Cancer cells were subjected to four different photosensitizer-mediated PDT and the resulting gene expression profiles were compared. We identified many differentially expressed genes reported previously as well as new genes for which the functionfunctions in PDT are still unclear. Our current results not only advance the general understanding of PDT but also suggest that distinct molecular mechanisms are involved in different photosensitizer-mediated PDT. Elucidating the signaling mechanisms in PDT will provide information to modulate the antitumor effectiveness of PDT using various photosensitizers. Keywords Cancer biology, drug toxicology, chemopreventation

Introduction Photodynamic therapy (PDT) is a novel medical procedure that holds considerable promise for the treatment of a variety of tumors.1 PDT is based on the light activation of photosensitizers that are selectively retained by proliferating tissues such as tumor cells in forming a concentration gradient against normal adjacent tissues.2 Photosensitizers when activated by light can produce reactive oxygen species (ROS) including singlet oxygen, superoxide anion, and hydroxyl radical. ROS can subsequently induce phototoxicity reaction in cancer cells. Following the onset of phototoxicity, the downstream cellular responses include inflammatory reaction, vascular damage, the formation of cytotoxic products, and, ultimately, apoptosis.3 Most photosensitizers are fluorescent so their subcellular localization after entering the cell can be determined using fluorescence microscopy.4 The intracellular localization of these photosensitizers has been reported previously5,6 (Table 1). Photosensitizers are taken up into cells by different pathways according

to their individual molecular structure, charge, and solubility (Table 1). 5-Aminolevulinic acid (5-ALA)induced endogenous photosensitization is a novel approach to both PDT and tumor detection that utilizes the heme biosynthetic pathway to produce endogenous porphyrins, particularly protoporphyrin IX, an effective photosensitizer. Excess exogenous 5-ALA can produce 1

Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan 2 Department of Otolaryngology, College of Medicine, National Taiwan University Hospital and National Taiwan University, Taipei, Taiwan 3 Biomedical Engineering Center, Industrial Technology Research Institute, Hsinchu, Taiwan 4 Department of Biological Science and Technology, China Medical University, Taichung, Taiwan Corresponding author: Ju-Hwa Lin, Department of Biological Science and Technology, China Medical University, No. 91 Hsueh-Shih Road, Taichung 404, Taiwan, Republic of China. Email: [email protected]

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Table 1. Structures and subcellular localization of photosensitizers. Photosensitizer

Structure

Subcellular localization

5-ALA

Mitochondria

mTHPC, Foscan1

Mitochondria, nuclear envelope, plasma membrane

TPPS2a

AlPcS2a

Lysosome, vesicle

Lysosome, vesicle, plasma membrane

5-ALA: 5-aminolevulinic acid; mTHPC: meta-tetrahydroxyphenylchlorin; TPPS2a: meso-tetraphenylporphyrin disulfonate; AlPcS2a: disulfonated aluminium phthalocyanine.

porphyrins that, when photoactivated, generate the photosensitizing effect for PDT.7 Porphyrins are first formed in the mitochondria and then rapidly diffuse to other intracellular membrane sites. Foscan is a promising second-generation photosensitizer. It appears to be among the most effective photosensitizers in PDT, requiring only very low drug doses (as little as 0.1 mg/ kg) and light doses (as low as10 J/cm2) for satisfactory efficacy.3 Previous publications reported that Foscan mainly localizes in the endoplasmic reticulum and mitochondria of human colon adenocarcinoma HT29 cells,8 while preferentially confines to the mitochondria and the perinuclear region of murine myeloid leukemia cells.9 Meso-tetraphenylporphyrin disulfonate (TPPS2a) differs only in side groups on the phenyl rings from the lipophilic photofrin, a widely used photosensitizer. TPPS2a is taken up initially into lysosomes via

endocytosis5 and induces apoptosis.10 Disulfonated aluminium phthalocyanine (AlPcS2a) is localized in vesicles suggestive of lysosomes,11 exhibiting additional advantages of being easily synthesized, inexpensive, comme rcially available, and composed of a single molecular species. Due to the differences in subcellular localization and the molecular mechanisms involved, the efficacy of photosynthesizers varies greatly with drug doses and light doses (Table 2). Differences in cell types, photosensitizers used, and incubation and illumination conditions can all significantly alter the outcome of PDT. For example, as mentioned above, Foscan is the most effective sensitizer in PDT studied so far. In vitro, Foscan is about 20-fold more effective in terms of drug dose than porphyrin derivatives in the treatment of HT29 colon adenocarcinoma cells, possibly because of the difference in drug uptake pathways as well as the presence of other inactive component/ components in porphyrin derivatives.12 Thus, no consistent model has emerged on the action of PDTinducing photosynthesizers in view of the very fragmented data from previous independent studies. In this study, we attempted to elucidate a consistent molecular model of PDT using an identical cell line to study the gene expression profiles of the four above-mentioned PDT-inducing photosynthesizers. Due to its capacity to affect a wide range of downstream cellular events, PDT appears to stimulate many different signaling pathways that may have either agnostic or antagonistic effects. For the apoptotic process, it has been shown that some of the pathways that contribute to the demise process are activated as well as others that antagonize cell death, so the ultimate survival of a given cell is determined by the combined action and/or interaction of these different pathways.13,14 To better understand, in a global view, the molecular mechanism/mechanisms involved in the response of tumor cells to PDT, microarray analysis was used to help identify genes that may contribute to the PDT response. Oligonucleotides-based microarrays have been shown to be a rapid and effective experimental instrument to monitor differential gene expressions.15 Over the last decade, array technologies have developed high-throughput means to identify molecular targets associated with biological and clinical phenotypes by comparing samples representative of distinct pathophysiological states. Gene expression profiling provided by microarray has been employed to define, at the molecular level, the clinical and histopathological phenotypes of given tumors.16 Expression


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Table 2. Drug treatment and photoirradiation of photosensitizers.a Photosensitizer

Photosensitizerpreincubated time (h)

5-ALA mTHPC TPPS2a

3 24 18

AlPcS2a

24

Lighting Cell time survival (%)

Light treatment 635 + 5 nm, LED red light with 55 mW/cm2 652 nm, diode laser light with 100 mW/cm2 430 nm (390–450 nm), a bank of four fluorescent tubes blue light with 7 mW/cm2 635 + 5 nm, LED red light with 55 mW/cm2

35 s 4s 28 s

65 + 5 66 + 5 60 + 5

14 min

69 + 5

5-ALA: 5-aminolevulinic acid; mTHPC: meta-tetrahydroxyphenylchlorin; TPPS2a: meso-tetraphenylporphyrin disulfonate; AlPcS2a: disulfonated aluminium phthalocyanine. a The uniformity of the illumination was confirmed by a phototoxicity assay.

profiling and pattern identification using various available microarray platforms are becoming novel tools for functional classification of genes and pathway discovery.16 To elucidate the signal transduction pathways in different photosensitizer-mediated PDT, we analyzed differential gene expression between untreated cultured Ca9-22 human gingival carcinoma cells and PDT-treated Ca9-22 cells with the use of oligonucleotides-based microarray technology. In this study, we focused on the similarities and differences in gene expression patterns generated after the treatment of four different photosensitizers and the changes in expression of the selected genes in time courses. To our knowledge, this study is the first report in conducting a large-scale survey of the variations in gene expression patterns in different photosensitizermediated PDT-treated cancer cells with a time course.

Materials and methods Cell culture Human gingival cancer (oral squamous cell carcinoma) cell line Ca9-22 was obtained from Japanese Collection of Research Bioresources cell bank. Stock culture of Ca9-22 cells was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin solution, and 10% fetal calf serum (FCS; Life Technologies Inc., Gaithersburg, MD, USA). Cell cultures were maintained at 37 C in a humidified atmosphere of 95% air and 5% carbon dioxide.

Photodynamic treatment The PDT groups of cells were preincubated for 3 h with 1 mM of 5-ALA (Sigma, St. Louis, MO, USA), 18 h with 0.2 mg/ml of TPPS2a(PCI Biotech, Lysaker,

Norway), 24 h with 20 mg/ml of AlPcS2a (Industrial Technology Research Institute, Chutung, Hsinchu, Taiwan), or 24 h with 0.2 mg/ml of metatetrahydroxyphenylchlorin (mTHPC, Foscan1; Biolitec Pharmaceuticals Ltd, Dublin, Ireland) in a culture medium containing 10% FCS (except for the 5-ALA group) in strictly subdued light conditions (1 mW/ cm2), respectively (Table 2). The cells were then irradiated separately in photosensitizer-free and phenol red-free medium. The light sources and treatment parameters are summarized in Table 2. After PDT treatment, the culture media was refreshed with complete media, and the cells were harvested after 30 min, 3 h, and 24 h for RNA extraction. The survival rates of the cells were about 65 + 5% at 24 h after PDT in all PDTtreated groups. Control groups consisted of cells that were not incubated with a photosensitizer and had no light treatment, the cells that were not incubated with a photosensitizer but had light treatment, and the cells that were incubated with a photosensitizer but without light treatment.

Assessment of photosensitizer-induced cytotoxicity on Ca9-22 Cells Ca9-22 cells were grown on 96-well plates at a density of 6000 cells/well overnight. The culture medium was removed and replaced with phenol red-free DMEM medium (150 ml/well) containing specific concentrations of photosensitizers as described above. The cells were incubated for a specific period with different photosensitizers with strictly subdued light conditions and then irradiated as mentioned above (Table 2). After light irradiation, the medium was replaced with DMEM containing 10% FCS. After 24 h, cell survival was measured using 3(4,5-dimethyl-thiazoyl-2-yl) 2,5 diphenyltetrazolium bromide assay. Each individual phototoxic experiment was repeated three times.


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RNA extraction Total cellular RNA was isolated from untreated and PDT-treated Ca9-22 cells using the RNeasy mini kit (Qiagen, Venlo, Netherlands), according to manufacturer’s instructions. The purity and integrity of RNA were assayed using GeneQuant™ pro RNA/DNA Calculator (Amersham Pharmacia Biotech, Amersham, UK) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California, USA). The absorbance ratio, A260/280, of 1.7–2.1 indicates that the quality of RNA is adequate to be applied in the array experiments.

Microarray procedures Total RNA of 2.5 mg from cells per time point and three controls from each of the four drug-treated samples were used in the microarray experiments. RNA samples were labeled using the Agilent’s Low RNA Input Fluorescent Linear Amplification kit (Agilent). Amplified RNA of 10 mg was used for each duplicate. Hybridization was carried out on Phalanx Human OneArray™ (HOA) microarray (version 1.0; Phalanx Biotech; http://www.phalanx.com.tw) at 50 C overnight. Following hybridization, the slides were washed for 15 min with 2 saline sodium citrate (SSC), 0.2% sodium dodecyl sulfate (SDS) at 42 C, 15 min at 42 C with 2 SSC, and 15 min at room temperature with 0.2 SSC consecutively. Duplicate chips were used for each RNA sample. Each array was scanned using the GenePix1 4000B microarray scanner (Axon Instruments Inc., Union City, California, USA) and analysis was carried out using GeneSpring software (Agilent, Santa Clara, California, USA).

Statistical analysis Data are presented as mean + SE. Levels of significance were calculated using analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons. A p value of