ANTICANCER RESEARCH 31: 763-770 (2011)
Anticancer Effects of Novel Photodynamic Therapy with Glycoconjugated Chlorin for Gastric and Colon Cancer MAMORU TANAKA1, HIROMI KATAOKA1, MOTOSHI MABUCHI1, SHIHO SAKUMA2, SATORU TAKAHASHI3, RYOTO TUJII4, HARUO AKASHI4, HIROMI OHI5, SHIGENOBU YANO5,6, AKIMICHI MORITA2 and TAKASHI JOH1
Departments of 1Gastroenterology and Metabolism, 2Dermatology, and 3Pathology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 4Research Institute for Natural Sciences, Okayama University of Science, Okayama, Japan; 5Endowed Research Section, Photomedical Science, Innovative Collaboration Center, Kyoto University, Kyoto, Japan; 6Graduate School of Material Science, Nara Institute of Science and Technology, Nara, Japan
Abstract. Background/Aim: Photodynamic therapy (PDT) is an attractive, minimally invasive modality for cancer therapy that utilizes the interaction of light and photosensitizer. To improve the efficacy of PDT, development of cancer specificity of the photosensitizer is needed. Cancer cells consume more glucose than normal cells. In this study, the efficacy of PDT using a newly developed photosensitizer, glycoconjugated chlorin (H2TFPC-SGlc), was compared with Talaporfin, which is clinically used in Japan. Materials and Methods: Photosensitizers were administered to gastric and colon cancer cell lines, followed by irradiation of light, and the cell death-inducing effects were compared. Xenograft tumor mouse models were established and photosensitizer accumulation was assessed and antitumor effects analyzed. Results: In vitro, H2TFPC-SGlc was 30 times more cytotoxic to cancer cells than was Talaporfin. In vivo, H2TFPC-SGlc accumulation was higher in xenograft tumors and significantly suppressed tumor growth when compared with Talaporfin. Conclusion: This novel glycoconjugated chlorin is potentially useful in PDT. Photodynamic therapy (PDT) is a promising non-invasive treatment for cancer (1-3). PDT involves the administration of a photosensitizer and visible light irradiation at a specific
Correspondence to: Hiromi Kataoka, MD, Ph.D., Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel: +81 528538211, Fax: +81 528520952, e-mail:
[email protected] Key Words: Photodynamic therapy, PDT, glycoconjugated chlorin, apoptosis, gastric cancer, colon cancer.
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wavelength to produce reaction by the photosensitizer (1). Activation of the photosensitizer leads to a conversion of molecular oxygen into various highly reactive oxygen species (ROS), which directly kill tumor cells or damage tumor-associated vasculature (1, 2). PDT has several advantages over other conventional cancer treatments (4). It is relatively non-invasive because irradiation is limited to the tumor site (5), and it shows lower systemic toxicity and relatively selective destruction of tumors, partly due to preferential localization of photosensitizer within the tumor (2). Thus, PDT has been widely employed against various tumors to which irradiation can be applied directly, such as lung, esophageal, gastric, breast, head and neck, bladder and prostate carcinomas (1). When compared with other therapies, PDT often produces higher cure and lower recurrence rates (6). However, as every technique has its limitations, PDT has been limited by the insufficient efficacy of photosensitizers. Despite being the most widely used in photosensitizer clinical settings, Photofrin, a first-generation photosensitizer, has several disadvantages, including long-term skin photosensitivity and a short absorption wavelength that limits tissue penetration (2, 5). Some second-generation photosensitizers have been shown to improve efficacy and reduce side-effects when compared with first-generation photosensitizers (5, 6). Talaporfin, a second-generation photosensitizer, has several advantages over first-generation photo sensitizers, such as reducing the photosensitization period and requiring a shorter interval between drug administration and laser light exposure as compared to photofrin (7). Talaporfin-mediated PDT has been examined in the treatment of several different type of solid tumor (4, 8). However, issues related to insufficient efficacy and skin photosensitivity remain unsolved; thus,
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ANTICANCER RESEARCH 31: 763-770 (2011) more effective photosensitizers are expected to be developed (3). Tumors consume higher levels of glucose than normal cells, a phenomenon known as the Warburg effect (9). Several glycoconjugated porphyrins have been synthesized and evaluated for photocytotoxicity (10), but there have been few reports on glycoconjugated chlorin. Yano and colleagues have developed several glycoconjugated chlorins and have reported excellent photocytotoxicity (11, 12). In this study, we evaluated the efficacy of PDT with a novel photosensitizer, glycoconjugated chlorin (H2TFPCSGlc), as compared with Talaporfin in vitro and in vivo.
Materials and Methods Photosensitizers. H2TFPC-SGlc [glycoconjugated chlorin; 5,10,15,20-tetrakis (4-(β-D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl)-2,3-(methano(N-methyl) iminomethano) chlorin] (Figure 1A) was prepared by the method described elsewhere (12). Talaporfin sodium (mono-l-aspartyl chlorin6, Laserphyrin®) was purchased from Meiji Seika (Tokyo, Japan). Cell culture. The human gastric cancer cell lines MKN28 (No. 0253; Japanese Cancer Research Resources Bank, Tokyo, Japan) and MKN45 (No. 0254; Japanese Cancer Research Bank, Tokyo, Japan) were cultured in RPMI1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% ampicillin and streptomycin. The human colon cancer cell lines HT29 (No. HTB-38; American Type Culture Collection, VA, USA) and HCT116 (No. CCL-247; American Type Culture Collection) were cultured in McCoy’s 5A Medium (Sigma-Aldrich) supplemented with 10% FBS and 1% ampicillin and streptomycin. Cells were cultured under an atmosphere of 5% CO2 at 37˚C. In vitro PDT. Gastric and colon cancer cells (MKN28, MKN45, HT29 and HCT116) were incubated with photosensitizer in culture medium for 24 h. Cells were washed once with, immersed in PBS, and irradiated at 16 J/cm2 (intensity: 37 mW/cm2) using an light emitting diode (LED) system (Omnilux PDT™; Omnilux, Cheshire, UK) emitting 633 nm. PBS in the wells was replaced with medium supplemented with 2% FBS, followed by incubation for a specified time before analyses. Cell viability assay. Cell viability was determined by WST-8 cell proliferation assay. Gastric and colon cancer cells were seeded into 96-well culture plates at 5×103 cells/100 μl/well and incubated overnight. Cells were incubated with photosensitizers at 37˚C for 24 h, irradiated and incubated for a further 24 h. To determine survival, cells were incubated with cell counting kit-8 (Dojindo, Kumamoto, Japan) for 4 h and absorption at 450 nm was measured with a microplate reader (SPECTRA MAX340; Molecular Devices, Silicon Valley, CA USA). Cell viability was expressed as a percentage vs. untreated control cells and the half maximal (50%) inhibitory concentration (IC50) was calculated. Flow cytometric analysis. In order to label apoptotic cells, cells were incubated with Active Caspase-3-FITC (BD Pharmingen™, Tokyo, Japan) at 4˚C for 30 min in the dark and were neutralized with binding buffer. Cells were then analyzed using a FACScantⅡ (BD Biosciences, Tokyo, Japan). Analyses were performed at 0, 1, 2, 4, 8, 12, 16, 20 and 24 h after PDT. At least 10,000 events were collected for each sample.
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Animals and tumor models. Pathogen-free female nude mice (BALB/c Slc-nu/nu) aged 4 weeks and weighing 20-25 g were obtained from Japan SLC (Kyoto, Japan). Animals were allowed to acclimatize for 2 weeks in the animal facility before any interventions were initiated. Xenograft tumor models were established by implanting 2×106 colon cancer cells (HT29, HCT116) in 200 μl of PBS subcutaneously. The procedures in these experiments were approved by the Nagoya City University Center for Experimental Animal Science, and mice were raised according to Nagoya City University for Animal Experiments. Spectrophotometric analysis. The accumulation of photosensitizers in the xenograft tumors was examined using a semiconductor laser with a VLD-M1 spectrometer (M&M Co., Ltd., Tokyo, Japan) that emitted a laser light with a peak wavelength of 405±1 nm and a light output of 140 mW. The spectrometer and its accessory software (BW-Spec V3. 24; B&W TEK, Inc., Newark, DE, USA) were used to analyze the spectrum waveform and revealed an amplitude peak (relative fluorescent intensity) at 505 nm for autofluorescence, at 655 nm for H2TFPC-SGlc and at 675 nm for Talaporfin. The relative intensities of the photosensitizers (with concentrations ranging from 0 to 50 μg/ml) measured by the spectrometer were observed to have a linear correlation with Talaporfin concentration. To reduce measurement error, the relative fluorescence intensity ratios of photosensitizers in the target tissue, which were calculated by dividing the relative fluorescence intensity by that of autofluorescence, were compared. In vivo PDT. When tumors grew to approximately 100 mm3, mice were given an intravenous injection (via tail vein) of photosensitizer (H2TFPC-SGlc or Talaporfin) at a dose of 6.25 μmol/kg. At 4 h after injection, tumors were illuminated with a 633 nm LED at a dose of 37.5 J/cm2. Tumor growth was monitored daily by measuring tumor volume with vernier calipers. Tumor volume was calculated using the following formula: (length × width × depth)/2. Results were analyzed by multiple testing (Holm method) between groups. Statistical analysis. Descriptive statistics and simple analyses were carried out using the statistical package R version 2.4.1 (www.rproject.org/). Antitumor effects were analyzed by the BonferroniHolm method. P-values of
