Photocytotoxicity, subcellular localization and apoptosis

Photodynamic effects of mTHPC on human colon adenocarcinoma cells: Photocytotoxicity, subcellular localization and apoptosis

Leung, W N

Photodynamic Effects of mTHPC on Human Colon Adenocarcinoma Cells: Photocytotoxicity, Subcellular Localization and Apoptosis(para)

ABSTRACT

The photodynamic properties of meta-tetra(hydroxyphenyl)chlorin (mTHPC), a promising second-generation photosensitizer, were investigated using a human colon adenocarcinoma cell line (Colo 201 cells). The study on photocytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5– diphenyltetrazolium bromide reduction assay showed that mTHPC was an effective photosensitizer on Colo 201 cells. The photocytotoxicity of mTHPC showed both drug and light dose-dependent characteristics. To reach LD^sub 50^, namely, the dose at which 50% of the cells were killed, only 0.45 +/- 0.15 (mu)g/mL of mTHPC and 3 J/cm^sup 2^ of light dose were required. The presence of 10% fetal calf serum in culture medium significantly decreased the incorporation of mTHPC into cells and resulted in the reduction of photodynamic efficacy. Using confocal laser scanning microscopy, mTHPC was first shown to localize in lysosomes rather than in mitochondria. Furthermore, nuclear stainings demonstrated that photodynamic therapy with mTHPC induced apoptosis in Colo 201 cells.

(para)Posted on the website on January 28, 2002.

Abbreviations: AMD, age-related macular degeneration; AO, acridine orange; BPD-MA, benzoporphyrin derivative monoacid ring A; Colo 201 cells, human colon adenocarcinoma cell line; EB, ethidium bromide; FCS, fetal calf serum; HPD, hematoporphrin derivatives LD^sub 50^, the dose at which 50% of the cells were killed; LSM, laser scanning microscopy; mTHPC, meta-tetra(hydroxyphenyl)chlorin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5– diphenyltetrazolium bromide; Npe6, mono-L-aspartyl chlorin e6; PBS, phosphate-buffered saline; PDT, photodynamic therapy; RPMI, Roswell Park Memorial Institute; R123, rhodamine 123.

INTRODUCTION

Photodynamic therapy (PDT) is an innovative cancer treatment modality in which a nontoxic photosensitive drug is preferentially absorbed or retained by malignant tissues after intravenous injection and then subsequently activated by localized light of an appropriate wavelength to induce cell death (1-3). The photodestruction of diseased tissues was considered to be mediated by the light-catalyzed formation of active oxygen species, notably singlet oxygen (2,3). The first clinical approval for PDT based on Photofrin(R) was obtained in 1993 in Canada for palliative treatment of oesophageal cancer and superficial bladder cancer-in response to the growing demand for a less debilitating, safer and more effective cancer treatment. By far, Photofrin-PDT has been approved by regulatory agencies in most developed countries for treatment of various cancer indications (1,4-7). PDT is also showing promising results for treatment of a variety of nonmalignant diseases, such as age-related macular degeneration (AMD), arteriosclerosis and psoriasis (2,4,8). Recently (in April 2000), PDT based on Visudyne(R) (namely, benzoporphyrin derivative monoacid ring A [BPD-MA]), one of the second-generation photosensitizers, has been approved by the U.S. Food & Drug Administration for the treatment of the wet form of AMD.

Because of the drawbacks of Photofrin, a number of new second-generation photosensitizers have been developed (1,9-11). meta-Tetra(hydroxyphenyl)chlorin (mTHPC) is a promising second-generation photosensitizer. The maximum absorption is at 652 nm (a wavelength that is more easily tumor penetrating) (1,4). This compound 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 as 10 J/cm^sup 2^) for efficacy. In comparison with Photofrin, mTHPC is about 100-200 times more effective in terms of the overall PDT dose (drug dose x light dose). Tumor to normal tissue ratios of 10-15 have been shown, and the duration of skin photosensitivity seems to be less than for Photofrin (12,13). mTHPC has been used to treat diverse complications (1,4,14). More recently (in June 2001), PDT with Foscan(R) (Temoporfin, mTHPC) has received approval from the European Medicines Evaluation Agency (EMEA) for the palliative treatment of patients with advanced head and neck squamous cell carcinoma, for whom earlier therapies have failed and for whom radiotherapy, surgery or systemic chemotherapy is unsuitable. Although mTHPC is generally considered as a potent sensitizer, many of the fundamental mechanisms of mTHPC-mediated PDT remain unclear. In this study the in vitro photodynamic efficacy of mTHPC in the human colon adenocarcinoma cell line (Colo 201 cells) was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of serum on its fluorescent properties and the photodynamic efficacy of the drug were also explored. Further, the intracellular localized sites of mTHPC were studied using confocal laser scanning microscopy (LSM) and costaining with organelle probes. Finally, the mode of cell death after PDT was identified by nuclear staining.

In conclusion, this study showed evidence that mTHPC was an effective photosensitizer on human colon cancer cells of Colo 201. The photocytotoxicity showed drug- and light– dependent characteristics. It was observed that the aggregated forms of mTHPC were preferentially taken up by Colo 201 cells and subsequently monomerized inside the cells. This study also demonstrated that mTHPC was localized in lysosomes of Colo 201 cells, and apoptosis was induced by mTHPC-PDT. Apart from mitochondria (15,36-39), lysosomes no doubt play an important role in PDT-mediated cell death (25,26,35,36), although most of the fundamental mechanisms of mTHPC-mediated PDT remain unclear. Finally, we can conclude that the subcellular localization of photosensitizers and the mode of cell death would be dependent on the drugs and cell lines used. The biochemical events of mTHPC-PDT-induced apoptosis and the response of tumor in the animal model deserve to be further investigated.

Acknowledgements-We thank Ms. L. Y. Man for technical assistance in confocal microscopy. We also would like to thank Prof. Stanley B. Brown for reviewing the manuscript. This work was supported by the RGC Earmarked Research Grant of the University Grants Committee of Hong Kong (RGC/97-98/59).

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W. N. Leung1, X. Sun*1,2, N. K. Mak2 and C. M. N. Yow2

1School of Chinese Medicine and 2Department of Biology, Hong Kong Baptist University, Hong Kong, People’s Republic of China

Received 20 November 2001; accepted 17 January 2002

*To whom correspondence should be addressed at: School of Chinese Medicine, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong, People’s Republic of China. Fax: 852-3411-2461; e-mail: awnleung@hkbu.edu.hk

Copyright American Society of Photobiology Apr 2002

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