Sonodynamic Antitumor Effect of Benzoporphyrin Derivative Monoacid Ring A on KLN205 Cells

Tomohiro Osaki, Motoshi Tajima, Yoshiharu Okamoto, Satoshi Takagi, Takeshi Tsuka, Tomohiro Imagawa, Saburo Minami

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DOI: 10.4236/jct.2011.22011                

Abstract

Sonodynamic therapy is a new cancer treatment based on the synergetic effect of ultrasound and a drug. In this study, ultrasonically induced antitumor effects of benzoporphyrin derivative monoacid ring A (BPD-MA) on KLN205 cells were investigated. KLN205 cells were irradiated at an ultrasonic frequency of 3 MHz with 10 μg/ml BPD-MA. The ultrasonically induced cell damage significantly increased as an ultrasonic intensity and ultrasound exposure time increased. Confocal microscopic examination revealed that the irradiated cells were induced chromatin condensation and phosphatidylserine exposure. The synergistic effect of the ultrasound exposure and BPD-MA on the tumor cell adhesion rate was significant.

Keywords

BPD-MA, Cell Damage, Sonodynamic Therapy, Sonosensitizer, Ultrasound

1. Introduction

Sonodynamic therapy (SDT) is a promising new method for cancer treatment utilizing the synergetic effect of ultrasound and a drug as sonosensitizer [1,2]. Ultrasound has some advantages, which includes an appropriate tissue attenuation coefficient for penetrating and reaching deep-seated tissues while maintaining the ability to focus energy onto a small volume. These unique properties could be available for noninvasive treatment of deepseated tumors compared to electromagnetic modalities, such as laser beams.

It has been reported that ultrasound energy enhanced the cytotoxicity of anticancer drugs [3-5]. However, these anticancer drugs have side effects. Therefore, it is necessary for establishment of anticancer effect to identify the effective sonosensitizer with strong cytotoxicity on tumor cells, few side effects, and high tumor-selectivity. The tumor-localizing porphyrins, which have been used as sensitizers in photodynamic therapy (PDT), have also been evaluated as a sonosensitizer [6]. In contrast to anticancer drugs, porphyrins are nontoxic without laser irradiation or ultrasonication. The side effects in surrounding normal tissues are minimized by the tumoraccumulative property of porphyrins and the geometrical selectivity by localized ultrasonic exposure.

Hematoporphyrin (Hp), aminolevulinic acid, and benzoporphyrin derivative monoacid ring A (BPD-MA) are used as a photosensitizer. Hematoporphyrin, one of the first generation photosensitizer, prolonged cutaneous photosensitivity of 1 - 3 months [7-9]. Benzoporphyrin derivative monoacid ring A (BPD-MA) is one of the second generation photosensitizers. The clearance from the body is more rapid than Hp, and skin photosensitivity lasts for only a few days [10,11]. This short clearance of photosensitizer can be a great advantage in treatment.

To our knowledge, the synergistic effects of ultrasound and BPD-MA on tumor cells were not reported. In this study, ultrasonically induced antitumor effects of BPD-MA on murine lung squamous cell carcinoma (KLN205) cells were investigated, in order to apply SDT with BPD-MA in a clinical setting. To estimate the efficacy of SDT, it was observed the cell adhesion rate and the morphological change of tumor cells following sonication with BPD-MA.

2. Materials and Methods

2.1. Cell Culture

KLN205 cells were obtained from the Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan). KLN205 cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), penicillin (100 IU/ml), and streptomycin (0.1 mg/ml) (Wako, Osaka, Japan) in a humidified incubator at 37˚C with a mixture of 5% CO₂ and 95% air. The cells were used for assays when they were in logarithmic growth. A total of 3 × 10⁵ cells were cultured in each 35-mm culture dish for 24 h. Culture dishes were divided randomly and evenly into 4 groups (control, ultrasound alone, BPD-MA alone and BPD-MA + ultrasound).

2.2. Photosensitizer

Liposomal BPD-MA was kindly donated by QLT Inc. (Vancouver, BC, Canada) in lyophilized powder form. Liposomal BPD-MA was reconstituted in sterile water (Fuso Pharmaceutical Industries, Ltd., Osaka, Japan) at a concentration of 2 mg/ml.

2.3. Ultrasound Exposure

Figure 1 shows a schematic diagram of the set up used in exposing KLN205 cells in vitro to ultrasound. Culture dishes were placed above a water bag filled with degassed waster. The transducer was placed under a water bag. Each gap between apparatuses was filled with echo gel (ITO physiotherapy & rehabilitation, Tokyo, Japan). Ultrasound generator US-700 (ITO physiotherapy & rehabilitation, Tokyo, Japan) was dual freuquency (1 and 3 MHz). The focus ultrasound transducer is a circular single disk in a diameter of 35 mm and a focal length of 22 mm.

Conflicts of Interest

The authors declare no conflicts of interest.

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