anticancer effects of novel photodynamic therapy with...

Abstract. Background/Aim: Photodynamic therapy (PDT)is an attractive, minimally invasive modality for cancertherapy that utilizes the interaction of light andphotosensitizer. To improve the efficacy of PDT, developmentof cancer specificity of the photosensitizer is needed. Cancercells consume more glucose than normal cells. In this study,the efficacy of PDT using a newly developed photosensitizer,glycoconjugated chlorin (H2TFPC-SGlc), was comparedwith Talaporfin, which is clinically used in Japan. Materialsand Methods: Photosensitizers were administered to gastricand colon cancer cell lines, followed by irradiation of light,and the cell death-inducing effects were compared. Xenografttumor mouse models were established and photosensitizeraccumulation was assessed and antitumor effects analyzed.Results: In vitro, H2TFPC-SGlc was 30 times more cytotoxicto cancer cells than was Talaporfin. In vivo, H2TFPC-SGlcaccumulation was higher in xenograft tumors andsignificantly suppressed tumor growth when compared withTalaporfin. Conclusion: This novel glycoconjugated chlorinis potentially useful in PDT.

Photodynamic therapy (PDT) is a promising non-invasivetreatment for cancer (1-3). PDT involves the administrationof a photosensitizer and visible light irradiation at a specific

wavelength to produce reaction by the photosensitizer (1).Activation of the photosensitizer leads to a conversion ofmolecular oxygen into various highly reactive oxygenspecies (ROS), which directly kill tumor cells or damagetumor-associated vasculature (1, 2). PDT has severaladvantages over other conventional cancer treatments (4). Itis relatively non-invasive because irradiation is limited to thetumor site (5), and it shows lower systemic toxicity andrelatively selective destruction of tumors, partly due topreferential localization of photosensitizer within the tumor(2). Thus, PDT has been widely employed against varioustumors to which irradiation can be applied directly, such aslung, esophageal, gastric, breast, head and neck, bladder andprostate carcinomas (1). When compared with othertherapies, PDT often produces higher cure and lowerrecurrence rates (6).

However, as every technique has its limitations, PDT hasbeen limited by the insufficient efficacy of photosensitizers.Despite being the most widely used in photosensitizerclinical settings, Photofrin, a first-generation photosensitizer,has several disadvantages, including long-term skinphotosensitivity and a short absorption wavelength that limitstissue penetration (2, 5).

Some second-generation photosensitizers have been shownto improve efficacy and reduce side-effects when comparedwith first-generation photosensitizers (5, 6). Talaporfin, asecond-generation photosensitizer, has several advantagesover first-generation photo sensitizers, such as reducing thephotosensitization period and requiring a shorter intervalbetween drug administration and laser light exposure ascompared to photofrin (7). Talaporfin-mediated PDT hasbeen examined in the treatment of several different type ofsolid tumor (4, 8). However, issues related to insufficientefficacy and skin photosensitivity remain unsolved; thus,

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Correspondence to: Hiromi Kataoka, MD, Ph.D., Department ofGastroenterology and Metabolism, Nagoya City UniversityGraduate 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.

ANTICANCER RESEARCH 31: 763-770 (2011)

Anticancer Effects of Novel Photodynamic Therapy withGlycoconjugated 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

0250-7005/2011 $2.00+.40

more effective photosensitizers are expected to be developed(3). Tumors consume higher levels of glucose than normalcells, a phenomenon known as the Warburg effect (9).Several glycoconjugated porphyrins have been synthesizedand evaluated for photocytotoxicity (10), but there have beenfew reports on glycoconjugated chlorin. Yano and colleagueshave developed several glycoconjugated chlorins and havereported excellent photocytotoxicity (11, 12).

In this study, we evaluated the efficacy of PDT with anovel photosensitizer, glycoconjugated chlorin (H2TFPC-SGlc), 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-tetrafluoro-phenyl)-2,3-(methano(N-methyl) iminomethano) chlorin] (Figure1A) was prepared by the method described elsewhere (12).Talaporfin sodium (mono-l-aspartyl chlorin6, Laserphyrin®) waspurchased 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 linesHT29 (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, HT29and HCT116) were incubated with photosensitizer in culture mediumfor 24 h. Cells were washed once with, immersed in PBS, and irradiatedat 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 cellproliferation assay. Gastric and colon cancer cells were seeded into96-well culture plates at 5×103 cells/100 μl/well and incubatedovernight. Cells were incubated with photosensitizers at 37˚C for24 h, irradiated and incubated for a further 24 h. To determinesurvival, cells were incubated with cell counting kit-8 (Dojindo,Kumamoto, Japan) for 4 h and absorption at 450 nm was measuredwith a microplate reader (SPECTRA MAX340; Molecular Devices,Silicon Valley, CA USA). Cell viability was expressed as apercentage vs. untreated control cells and the half maximal (50%)inhibitory concentration (IC50) was calculated.

Flow cytometric analysis. In order to label apoptotic cells, cells wereincubated with Active Caspase-3-FITC (BD Pharmingen™, Tokyo,Japan) at 4˚C for 30 min in the dark and were neutralized with bindingbuffer. Cells were then analyzed using a FACScantⅡ (BD Biosciences,Tokyo, Japan). Analyses were performed at 0, 1, 2, 4, 8, 12, 16, 20 and24 h after PDT. At least 10,000 events were collected for each sample.

Animals and tumor models. Pathogen-free female nude mice(BALB/c Slc-nu/nu) aged 4 weeks and weighing 20-25 g wereobtained from Japan SLC (Kyoto, Japan). Animals were allowed toacclimatize for 2 weeks in the animal facility before anyinterventions were initiated. Xenograft tumor models wereestablished by implanting 2×106 colon cancer cells (HT29,HCT116) in 200 μl of PBS subcutaneously. The procedures in theseexperiments were approved by the Nagoya City University Centerfor Experimental Animal Science, and mice were raised accordingto Nagoya City University for Animal Experiments.

Spectrophotometric analysis. The accumulation of photosensitizersin the xenograft tumors was examined using a semiconductor laserwith a VLD-M1 spectrometer (M&M Co., Ltd., Tokyo, Japan) thatemitted a laser light with a peak wavelength of 405±1 nm and alight output of 140 mW. The spectrometer and its accessorysoftware (BW-Spec V3. 24; B&W TEK, Inc., Newark, DE, USA)were used to analyze the spectrum waveform and revealed anamplitude peak (relative fluorescent intensity) at 505 nm forautofluorescence, at 655 nm for H2TFPC-SGlc and at 675 nm forTalaporfin. The relative intensities of the photosensitizers (withconcentrations ranging from 0 to 50 μg/ml) measured by thespectrometer were observed to have a linear correlation withTalaporfin concentration. To reduce measurement error, the relativefluorescence intensity ratios of photosensitizers in the target tissue,which were calculated by dividing the relative fluorescence intensityby that of autofluorescence, were compared.

In vivo PDT. When tumors grew to approximately 100 mm3, micewere given an intravenous injection (via tail vein) of photosensitizer(H2TFPC-SGlc or Talaporfin) at a dose of 6.25 μmol/kg. At 4 h afterinjection, tumors were illuminated with a 633 nm LED at a dose of37.5 J/cm2. Tumor growth was monitored daily by measuring tumorvolume with vernier calipers. Tumor volume was calculated usingthe following formula: (length × width × depth)/2. Results wereanalyzed by multiple testing (Holm method) between groups.

Statistical analysis. Descriptive statistics and simple analyses werecarried out using the statistical package R version 2.4.1 (www.r-project.org/). Antitumor effects were analyzed by the Bonferroni-Holm method. P-values of <0.05 were considered statisticallysignificant.

Results

PDT with H2TFPC-SGlc induced cell death in gastric andcolon cancer cell lines. We evaluated the cell death inducedby PDT with H2TFPC-SGlc. Cells were loaded withphotosensitizer (H2TFPC-SGlc or Talaporfin) for 24 h,irradiated with 633-nm LED light at 16 J/cm2, and incubatedfor 24 h. As shown in Figure 1B, PDT with H2TFPC-SGlcinduced cell death; however, H2TFPC-SGlc alone and lightalone did not cause any significant toxicity in MKN28 cells.Similar results were obtained with the other cell lines(MKN45, HT29 and HCT116) (data not shown). We thendetermined the IC50 at 24 h after PDT. PDT with H2TFPC-SGlc induced cell death about 30 times more efficiently inall cell lines, as compared to PDT with Talaporfin (Table Ⅰ).


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PDT with H2TFPC-SGlc induced apoptosis via ROSproduction. We investigated the mode of cell death inducedby H2TFPC-SGlc-mediated PDT. Cells were incubatedwith 1 μM photosensitizer (H2TFPC-SGlc or Talaporfin)for 24 h, followed by irradiation with 633-nm LED (16J/cm2). We measured the mean fluorescence intensity ofactive caspase-3 as a marker for apoptosis by FACS. Anincrease in mean fluorescence intensity was observed after4 h, and this increase continued, until peaking at 16 h(Figure. 1C). We then examined ROS induction by PDTwith H2TFPC-SGlc. Cells were incubated with 1 μMphotosensitizer (H2TFPC-SGlc or Talaporfin) for 24 h,followed by irradiation with 633-nm LED (16 J/cm2).Figure 1C shows the data for MKN45 cells. The same trendwas seen for the other cell lines (MKN28, HT29 andHCT116) (data not shown).

Accumulation of H2TFPC-SGlc in xenograft tumor of coloncancer. We examined whether photosensitizer (H2TFPC-SGlc or Talaporfin) accumulated in the xenograft tumorsestablished by subcutaneously implanting colon cancer cells(HT29 and HCT116). Four hours after intravenous injection

Tanaka et al: Novel PDT with Glycoconjugated Chlorin

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Figure 1. Cell death by photodynamic therapy. A: Chemical structure of H2TFPC-SGlc. B: Cells were loaded with 1 μM H2TFPC-SGlc for 24 h, andirradiated with 633 nm LED light at 16 J/cm2. Phase-contrast images were obtained by microscopy (original magnification, ×200). C: Cells wereloaded with 1 μM H2TFPC-SGlc for 24 h, and irradiated with 633 nm LED light at 16 J/cm2. Cells were them stained with active caspase-3-FITCand analyzed for apoptotic cells by FACS.

Table I. IC50 of the efficiency of photodynamic therapy.

IC50 (μM)

Gastric cancer Colon cancer

MKN28 MKN45 HT29 HCT116

Talaporfin 12.7 18.6 25.8 12.9H2TFPC-SGlc 0.56 0.48 0.52 0.35

(via tail vein) of photosensitizer (H2TFPC-SGlc orTalaporfin) at a dose of 6.25 μmol/kg, the spectrumwaveform from the xenograft tumors was analyzed by aspectrometer (VLD-M1). The spectrum waveform showedtwo peaks of fluorescence emission: one at 505 nm,corresponding to autofluorescence, and the othercorresponding to photosensitizers (H2TFPC-SGlc at 655 nm,and Talaporfin at 670 nm) (Figure 2A). Next, we measuredthe relative fluorescence intensity ratio of photosensitizers intumors and normal tissue surrounding the tumor using aspectrometer. The relative fluorescence intensity ratios ofboth photosensitizers were highest at 4 h after drug

administration (Figure 2B). H2TFPC-SGlc accumulated inthe tumor tissue to a significantly higher extent than in thenormal tissue surrounding the tumor.

Antitumor effects of H2TFPC-SGlc in vivo. In order toexamine the effects of H2TFPC-SGlc-mediated PDT ontumors in vivo, PDT was performed on xenograft tumormodels established by subcutaneously implanting colon cancercells (HT29, HCT116). When tumors grew to 75-150 mm3,mice were given an intravenous injection (via tail vein) ofphotosensitizer (H2TFPC-SGlc or Talaporfin) at a dose of 6.25 μmol/kg, followed by irradiation with a 633-nm LED


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Figure 2. Accumulation of H2TFPC-SGlc in tumors. A: Cells were inoculated in dorsal skin at a density of 5×106 cells/200 μl in PBS. Tumor-bearing mice were given intravenous injections of 6.25 μmol/kg photosensitizer (H2TFPC-SGlc or Talaporfin). Four hours after photosensitizeradministration, the spectrum waveforms of the tumor (left) and normal (right) tissue were analyzed using a VLD-M1 spectrometer. B: The relativeintensity of photosensitizer and autofluorescence was measured using a spectrometer at different times after injection. The relative fluorescenceintensity ratio in tumors and normal tissue was calculated by dividing the relative fluorescence intensity of photosensitizer by that of autofluorescence(left, tumor; right, normal tissue).

light (37.5 J/cm2). The xenograft tumors showed no changesin size immediately after PDT. However, at 48 h after PDT,the tumors were reduced in size. Little damage was observedin normal tissues surrounding the tumor (Figure 3A). Figure3A shows mice injected with in HCT116 cells. Similar resultswere obtained with the other cell line, HT29 (data not shown).As shown in Figure 3B, the tumor volume increased by about3-fold within 5 days in control mice (i.e. kept in darknesswithout photosensitizer administration, n=8), mice exposed tolight alone (i.e. illuminated without photosensitizeradministration, n=4) and mice exposed to H2TFPC-SGlc alone(i.e. administered H2TFPC-SGlc and kept in darkness, n=4).In contrast, tumor growth was significantly suppressed afterH2TFPC-SGlc-mediated PDT (n=8, p<0.01) and Talaporfin-

mediated PDT (n=8, p<0.01). H2TFPC-SGlc-mediated PDTwas more potent than that of Talaporfin-mediated PDT inxenograft tumor models (HT29 tumors, p<0.01; HCT116tumors, p<0.01) (Figure 3B). H2TFPC-SGlc-mediated PDTresulted in continued decreases in tumor volume for 5 dayspost-treatment.

Discussion

H2TFPC-SGlc was developed as a chlorine-basedphotosensitizer, and was expected to have a number ofadvantages, including significant reductions in darkcytotoxicity, improved water-solubility, greater cellularuptake, and sugar-dependent photocytoxicity (11-13). We


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Figure 3. Inhibition of tumor growth by photodynamic therapy with H2TFPC-SGlc. A: Tumor-bearing mice were given intravenous injections of6.25 μmol/kg photosensitizer (H2TFPC-SGlc or Talaporfin) and after 4 h of illuminated under 633 nm LED light (37.5 J/cm2). Tumor-bearingregions in mice treated with H2TFPC-SGlc-mediated PDT were photographed immediately and at 48 h after PDT. B: Relative tumor volumes weremonitored every day for 5 days in control mice (no treatment), and mice treated with light alone, H2TFPC-SGlc alone, H2TFPC-SGlc-mediatedPDT or Talaporfin-mediated PDT. Data are means±SD. Significance was determined by the Bonferroni-Holm method. P-values are **<0.01 (left,HT29 cells; right, HCT116 cells).

investigated whether H2TFPC-SGlc could act as a potentialphotosensitizer of PDT in gastric and colon cancer in vitroas well as in vivo. In vitro, H2TFPC-SGlc-mediated PDT isable to induce apoptosis and is about 30 times morecytotoxic than Talaporfin-mediated PDT. In xenograft tumormodels, H2TFPC-SGlc-mediated PDT suppressed tumorgrowth and had no adverse effects on surrounding tissues, ascompared to light alone, H2TFPC-SGlc alone and Talaporfin-mediated PDT. Our results indicate that PDT with H2TFPC-SGlc offers a minimally invasive therapeutic modality forclinical treatment of gastric and colon cancer.

Many experiments have been performed in order to developnew photosensitizers that show preferential accumulationwithin the target tumor tissue for various active targetingapproaches, such as peptide conjugates and antibodies (14-18),incorporation within liposomes (19, 20), and encapsulationwithin polymeric nanoparticles (21-26). For accumulation inthe target tumor, H2TFPC-SGlc was developed by linkingglucose to the photosensitizer chlorin. The uptake of H2TFPC-SGlc is greater than that of Talaporfin both in vitro (Sakuma etal., in submission) and in vivo (Figure 2A). These resultsindicate that the glucose-linked photosensitizer is a very usefuldrug delivery system for cancer cells.

Talaporfin has frequently been reported to shorten the invivo retention that increases the risk of phototoxicity and theinterval required between drug administration and lasertreatment (27). The optimal interval length of H2TFPC-SGlcwas 4 h, which is the same as that of Talaporfin (Figure 2B).These findings suggest that H2TFPC-SGlc-mediated PDThas the advantage of prolonged photosensitization, similarlyto Talaporfin.

In cancer therapy, intense research has been performed toidentify the molecules that regulate cross-talk betweenapoptosis and other major cell death subroutes (e.g. necrosisand autophagic cell death). The necrotic pathway is initiatedby photodamage to the endoplasmic reticula/Golgi body, theapoptotic pathway by photodamage to the mitochondria, andthe autophagic pathway also by photodamage to theendoplasmic reticula (28). We observed that PDT inducedapoptotic cell death with H2TFPC-SGlc in human gastric andcolon cancer cells. Apoptosis was also observed in byH2TFPC-SGlc-mediated PDT (Figure 1C). In humanmelanoma cells (COLO679), the subcellular localization ofH2TFPC-SGlc was investigated using fluorescence probes forintracellular organelles, and H2TFPC-SGlc was found toaccumulate in mitochondria, lysosomes, Golgi bodies andendoplasmic reticula (Sakuma et al., in submission). The sametrend was seen in with human gastric cancer cell lines (datanot shown), and it remains possible that PDT with H2TFPC-SGlc induces cell death via necrosis and/or autophagy.

PDT with H2TFPC-SGlc showed significant antitumoreffects in vitro (Table Ⅰ) and in vivo (Figure 3B). Talaporfin, asecond-generation photosensitizer, shows improved efficacy

when compared with first-generation photosensitizers (7). Inthis study, H2TFPC-SGlc-mediated PDT was more effectivethan Talaporfin-mediated PDT. PDT with H2TFPC-SGlcshowed significant antitumor effects using relatively low lightdoses (37.5 J/cm2). Several photosensitizers, includingPhotofrin and Talaporfin, have typically been used with over100 J/cm2 for carcinoma xenografts (3, 29-32), and low-doselight irradiation may reduce side-effects related to damage ofadjacent normal tissue.

In conclusion, we demonstrated that H2TFPC-SGlc-mediated PDT effectively suppresse the growth of xenografttumors, inducing apoptosis. In addition, H2TFPC-SGlc hadsuperior cancer cell selectivity and was able to reduce side-effects, such as prolonged skin phototoxicity. Based on thepotential and characteristics of H2TFPC-SGlc presented inthis study, we confirm that H2TFPC-SGlc is a potentialphotosensitizer for PDT in gastric and colon cancer.

Acknowledgements

This work was supported by a Japanese Foundation for Researchand Promotion of Endoscopy Grant, 2010, by a Grant from theMinistry of Education, Culture, Sports, Science, and Technology,Japan, and by Dr. Masamichi Ipponmatsu (Renaissance EnergyInvestment Co., Ltd., Kyoto, Japan). We would also like to thankYukimi Ito for technical assistance.

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Received December 26, 2010Revised February 24, 2011

Accepted February 25, 2011


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