akihisa kato1, hiromi kataoka1*, shigenobu yano2, kazuki ... · 3/14/2017 · translational...

1

Maltotriose conjugation to a chlorin derivative enhances the antitumor effects of

photodynamic therapy in peritoneal dissemination of pancreatic cancer

Akihisa Kato1, Hiromi Kataoka1*, Shigenobu Yano2, Kazuki Hayashi1, Noriyuki

Hayashi1, Mamoru Tanaka1, Itaru Naitoh1, Tesshin Ban1, Katsuyuki Miyabe1, Hiromu

Kondo1, Michihiro Yoshida1, Yasuaki Fujita1, Yasuki Hori1, Makoto Natsume1, Takashi

Murakami3, Atsushi Narumi4, Akihiro Nomoto5, Aya Naiki-Ito6, Satoru Takahashi6,

Takashi Joh1

1 Department of Gastroenterology and Metabolism, Nagoya City University Graduate

School of Medical Sciences, Nagoya, Japan

2 Graduate School of Materials Science, Nara Institute of Science and Technology,

Ikoma, Nara, Japan

3 Laboratory of Tumor Biology, Takasaki University of Health and Welfare, Takasaki,

Japan

4Department of Organic Materials Science, Graduate School of Organic Materials

Science, Yamagata University, Yonezawa, Japan

5Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture

on October 17, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 14, 2017; DOI: 10.1158/1535-7163.MCT-16-0670

http://mct.aacrjournals.org/

2

University, Osaka, Japan

6 Department of Experimental Pathology and Tumor Biology, Nagoya City University

Graduate School of Medical Sciences, Nagoya, Japan

Running title: PDT with Mal3-chlorin for disseminated pancreatic cancer

Keywords: photodynamic therapy, PDT, pancreatic cancer, peritoneal dissemination,

maltotriose-conjugated chlorin

Financial Information

This study was partially supported by JSPS KAKENHI grant numbers 26460947, the

Translational Research Network Program of the Japan Agency for Medical Research

and Development, AMED, 2015-2016 (to H. Kataoka), JSPS KAKENHI grant numbers

19350031, 25288028, the Japan–German Exchange Program supported by the JSPS and

the Deutsche Forschungsgemeinschaft (DFG) (to S. Yano) and Pancreas Research

Foundation of Japan (to A. Kato).

Contact Information




3

*Corresponding author: Hiromi Kataoka, MD, PhD. Department of Gastroenterology

and Metabolism, Nagoya City University Graduate School of Medical Sciences,

1-Kawasumi, Mizuho-cho, Mizuho-ku 467-8601, Nagoya, Japan. Tel: +81-52-853-8211,

Fax: +81-52-852-0952, E-mail: [email protected]

Conflict of interest statement: There are no potential conflicts of interest.

Abstract

Peritoneal dissemination is a major clinical issue associated with dismal prognosis and

poor quality of life for patients with pancreatic cancer; however, no effective treatment

strategies have been established. Herein, we evaluated the effects of photodynamic

therapy (PDT) with maltotriose-conjugated chlorin (Mal3-chlorin) in culture and in a

peritoneal disseminated mice model of pancreatic cancer. The Mal3-chlorin was

prepared as a water-soluble chlorin derivative conjugated with four Mal3 molecules to

improve cancer selectivity. In vitro, Mal3-chlorin showed superior uptake into

pancreatic cancer cells compared with talaporfin, which is clinically used. Moreover,

the strong cytotoxic effects of PDT with Mal3-chlorin occurred via apoptosis and

reactive oxygen species generation, whereas Mal3-chlorin alone did not cause any




4

cytotoxicity in pancreatic cancer cells. Notably, using a peritoneal disseminated mice

model, we demonstrated that Mal3-chlorin accumulated in xenograft tumors and

suppressed both tumor growth and ascites formation with PDT. Further, PDT with

Mal3-chlorin induced robust apoptosis in peritoneal disseminated tumor, as indicated by

immunohistochemistry. Taken together, these findings implicate Mal3-chlorin as a

potential next-generation photosensitizer for PDT and the basis of a new strategy for

managing peritoneal dissemination of pancreatic cancer.

Introduction

Pancreatic cancer is a highly aggressive disease with a dismal prognosis. The reported

5-year overall survival rate of patients with pancreatic cancer is less than 5% (1), due

mainly to the frequently advanced stage at diagnosis, rapid tumor growth, and

metastasis to distant organs such as the liver, lung and peritoneum (2). Peritoneal

dissemination of pancreatic cancer poses significant difficulties for both patients and

clinicians because the associated poor general condition of affected patients and

problems in assessing scattered tumors due to ascites, jaundice and ileus hamper the

administration of standard treatment. The treatment of choice for patients with

peritoneal dissemination is palliative systemic chemotherapy; however, its impact on the




5

primary pancreatic cancer is limited (3, 4). Thus, new methods of treating peritoneal

dissemination are needed.

One such approach, photodynamic therapy (PDT), is a promising, minimally invasive

modality for cancer therapy that utilizes reactive oxygen species (ROS) generated by the

photochemical reactions between a photosensitizer and irradiation (5, 6). The anticancer

effects of PDT are consequences of a low-to-moderately selective degree of

photosensitizer taken up by malignant cells, direct cytotoxicity due to ROS, and severe

tumor vascular damage that impairs blood supply to the treated area (7-9). PDT also has

several advantages over conventional radiation and chemotherapy with respect to side

effects and damage to normal tissue.

PDT using photofrin, a first-generation photosensitizer, is widely used clinically (10,

11). More recently, PDT using a second-generation photosensitizer, talaporfin, has

shown several advantages over PDT with photofrin including deeper penetration into

tissue and less prolonged photosensitization (12, 13), and has already been used to treat

a wide range of cancers (14-16). Nevertheless, PDT issues related to insufficient

efficacy and skin photosensitivity remain unsolved, and the need remains for

photosensitizers with better cancer cell selectivity.

We previously reported that PDT using a newly developed photosensitizer,




6

glucose-conjugated chlorin (G-chlorin), exerted high photocytotoxicity compared with

PDT with talaporfin (17, 18). G-chlorin with its four glucose molecules,

(5,10,15,20-tetrakis-(pentafluorophenyl)-2,3-[methano(N-methyl)iminomethano]

chlorin) is likely to elicit high photocytotoxicity in cancer cells due to their tendency to

accumulate glucose, called the Warburg effect (19). Unfortunately, G-chlorin is

insoluble in water, making it less suitable for clinical use. Furthermore, good water

solubility ensures rapid clearance from the body, avoiding cutaneous phototoxicity.

We have therefore developed a straightforward strategy to improve the water-solubility

of chlorin derivatives by using a oligosaccharide, such as maltotriose (Mal3), called

Mal3-chlorin and have reported its highly hydrophilicity and photocytotoxicity (20). In

the present study, we investigated the efficacy of PDT with a novel photosensitizer,

Mal3-chlorin, in culture and in a mouse model of disseminated peritoneal pancreatic

cancer using in vivo fluorescence imaging to monitor sequentially.

Materials and Methods

Photosensitizers

Talaporfin sodium (mono-l-aspartyl chlorin6, Laserphyrin®) was purchased from Meiji

Seika (Tokyo, Japan) (Figure 1A). Mal3-chlorin




7

(5,10,15,20-tetrakis-[4-(β-D-maltotriosylthio)-2,3,5,6-tetrafluorophenyl]-2,3-[methano-(

N-methyl)iminomethano]chlorin) was synthesized (Figure 1B) and provided by

Yamagata University (Yamagata, Japan) (20).

Cell culture

Human pancreatic cancer cell lines, AsPC1 and BxPC3, were obtained from the

American Type Culture Collection (ATCC, Rockville, MD) on Dec 3, 2015, while the

luciferase-transfected AsPC1/luc and BxPC3/luc lines were obtained from the JCRB

cell bank on Dec 1, 2015. Our cell lines were authenticated with STR-PCR profiling at

the cell banks and revealed to be not misidentified with any other cell lines. These cell

lines were certified to be free of fungal, bacterial and mycoplasmal contaminations by

the cell banks, and were distributed to our institution. All cell lines were cultured in

RPMI1640 medium (Wako Pure Chemical Industries Co. Ltd., Osaka, Japan)

supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37°C in 5%

CO2 humidified air, and experiments were carried out within 3 months of resuscitation.

Flow cytometric analysis

Pancreatic cancer cells were incubated with talaporfin (5 μM) or Mal3-chlorin (5 μM)

for 4 h and washed once with phosphate-buffered saline (PBS). Cells were then

harvested using 0.25% trypsin-EDTA (GIBCO) and analyzed using a FACS Canto II




8

Analyzer (BD Biosciences; excitation; 405 nm, emission; 660 nm). At least 10,000

events were collected for each sample.

In vitro PDT

Pancreatic cancer cells (AsPC1, BxPC3, AsPC1/luc and BxPC3/luc) were incubated

with photosensitizer in culture medium for 4 h. Cells were washed once with PBS,

covered with PBS, and irradiated at 13.9 J/cm2 (intensity: 30.8 mW/cm2) of LED light

(Opto Code Corporation), which emits 660 nm wavelength. Following irradiation, the

PBS in the wells was exchanged for medium supplemented with 10% FBS, and the cells

were incubated for the specified times before analyses.

Cell viability assay

Cell viability was analyzed by the WST-8 cell proliferation assay, with 5 × 103

cells/well of AsPC1, BxPC3, AsPC1/luc, and BxPC3/luc incubated with photosensitizer

for 4 h, and then irradiated as described in the preceding section. After incubation for 24

h, cells were incubated for 2 h with the Cell Counting Kit-8 (Dojindo, Kumamoto,

Japan) and absorption was measured with a microplate spectrophotometer (SPECTRA

MAX340; Molecular Devices, Kenilworth, NJ). Cell viability was expressed as a

percentage of untreated control cells and the half maximal (50%) inhibitory

concentration (IC50) was calculated.




9

Caspase 3/7 assay

Apoptosis in the AsPC1/luc cells was assessed using the Caspase-Glo3/7 Assay Kit

(Promega). AsPC1/luc cells were treated with photosensitizers (0.25 - 1 μM) at 37°C for

4 h, and irradiated. Analyses were performed at 4 h after PDT by adding 100 μl of

caspase-3/7 reagent to each well, mixing, and then incubating for 1 hour at room

temperature. Luminescence was measured using a Lu mat LB 9507 instrument (EG&G

BERTHOLD) and the data were normalized against the average value of non-treated

cells. Relative luminescence units (RLU) of the caspase 3/7 activity are expressed as

mean ± SD.

Estimation of cellular ROS production

AsPC1/luc cells were treated with or without Mal3-chlorin (0.25-1 μM) for 4 h with or

without subsequent irradiation (13.9 J/cm2). At 1 h after irradiation, cells were

incubated with 100 μg/ml 2’,7’-dichlorofluorescein-diacetate (DCFH-DA, Sigma) at

37°C for 20 min, in the dark. The cells were washed two times with warm PBS and

viewed with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The

fluorescence intensity scores of imaged cells were evaluated using the associated

software (21). Furthermore, H2O2 released into media collected from AsPC1/luc cells

with or without PDT was determined using ROS-GloTM H2O2 assay (Promega). Cells




10

were incubated with each photosensitizer (0.25-2 μM) for 4 h and irradiated. At 1 h after

PDT, cells were incubated in a H2O2 substrate solution at 37°C for 3 h and then luciferin

detection reagent at room temperature for 1 h. Luminescence was measured using a Lu

mat LB 9507 instrument (EG&G BERTHOLD) and the RLU of H2O2 concentrations

are expressed as mean ± SD, relative to the average value of non-treated cells.

Animals and peritoneal dissemination model of pancreatic cancer

Female nude mice (BALB/c Slc-nu/nu) aged 4 weeks and weighing 15-20 g were

obtained from Japan SLC and acclimated to the laboratory for 1 week. They were

housed at five animals per cage on pulp-chip bedding in an air-conditioned animal room

at 22 ± 2°C and 55 ± 5% humidity. All mice were maintained under specific

pathogen-free conditions with a 12-h light/dark cycle. To prepare the in vivo peritoneal

dissemination model, AsPC1/luc cells were injected intraperitoneally (i.p.) with 1 × 106

cells in 200 μl PBS. All animal experiments were performed under protocols approved

by the Institutional Animal Care and Use Committee of Nagoya City University

Graduate School of Medical Sciences.

In vivo spectrophotometric analysis

The accumulation of photosensitizer in the in vivo models was examined by

optical-filter imaging (Longpass Filter VL0470, cut-on; 470 nm, Asahi Spectra Inc.)




11

and using a semiconductor laser with a VLD-M1 spectrometer (M&M Co., Ltd.) 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.),

used to analyze the spectrum waveform, revealed an amplitude peak (relative

fluorescent intensity) at 505 nm for autofluorescence and 655 nm for photosensitizers.

The relative intensities of the photosensitizers were also measured

spectrophotometrically. To reduce measurement error, we divided the relative

fluorescence intensity by the autofluorescence intensity to compare relative

fluorescence intensity ratios of the tested photosensitizers in the target tissue.

In vivo PDT and bioluminescence imaging

One week after injection of the cells, bioluminescence was measured using a

multi-functional in vivo imaging system (IVIS; MIIS, Molecular Devices Japan) to

confirm the cell implantations. The mice were randomly allocated to three groups. Then,

the mice were given an i.p. injection with photosensitizer (talaporfin and Mal3-chlorin)

at 1.25 μmol/kg in saline. Four hours later, the mice were irradiated with 660-nm LED

light (Opto Code Corporation) at a dose of 13.9 J/cm2. This light device was placed just

above the abdomen so that the irradiation spot on the abdominal skin was 1.77 cm2

(diameter 1.5 cm), thus covering the lower abdomen and avoiding the liver. Treatment




12

was performed on day 0 and 7, as shown in Figure 4A. To visualize the peritoneal

tumors in mice, IVIS was performed as follows. The mice were anesthetized and

subsequently i.p. injected with D-luciferin (150 μg/g body weight, Wako, Ltd.) in

D-PBS. Two minutes later, a monochrome photograph was acquired followed

immediately by bioluminescence acquisition for 20 sec and in vivo fluorescence

imaging. From this, total luminescence flux from the region of tumor on day 0, 7, 14

was quantified using MetaMorph-MIIS software (Molecular Devices Japan) to monitor

tumor growth. The bioluminescence images were exported as false-colored images

using matched visualization scales. At the end of the experiment on day 21, mice were

sacrificed and ascites volumes were measured. Another in vivo experiment was also

conducted on the mice peritoneal tumor tissue to analyze apoptosis, whereby PDT with

each photosensitizer was administered only on day 14 followed by excision of the tumor

tissue on day 15 and fixation in 10% phosphate-buffered formalin.

Immunohistochemical analysis of apoptosis

For immunohistochemistry, the sections were immunostained as previously described

(22). In brief, apoptotic cells were detected by terminal deoxy nucleotidyl

transferase-mediated dUTP nick end labeling (TUNEL) assay as well as cleaved caspase

3 immunohistochemistry. TUNEL assays were performed using an In Situ Apoptosis




13

Detection Kit from Takara (Otsu, Japan). For detection of cleaved caspase 3,

deparaffinized sections of tumor from mice peritoneum were incubated with 1:100

diluted cleaved caspase 3 antibody (Cell Signaling Technology). Antibody binding was

visualized by a conventional immunostaining method using an autoimmunostaining

apparatus (HX System, Ventana, Tucson, AZ).

Statistical analysis

The statistical significance of differences was determined using Dunnett’s test,

Student’s t test, or the χ2 test as appropriate. Differences were considered statistically

significant at P < 0.05. Data are expressed as mean ± SD.

Results

Pancreatic cancer cells take up Mal3-chlorin more efficiently than talaporfin

We first examined the uptake of talaporfin and Mal3-chlorin in AsPC1/luc cells using

flow cytometric analysis, and then measured intensity of the characteristic red

fluorescence at the single cell level. The cells treated with Mal3-chlorin showed a

stronger signal (mean intensity: 1545) than both the non-treated cells (mean intensity:

44) and the talaporfin-treated cells (mean intensity: 80), indicated that more

Mal3-chlorin was taken up by cancer cells compared to talaporfin (Figure 1C).




14

The high cytotoxic effects of PDT with Mal3-chlorin on pancreatic cancer cells

were induced through apoptosis and ROS generation

We next evaluated the cell death induced by PDT with Mal3-chlorin. As shown in

Figure 2A, PDT with Mal3-chlorin induced cell death. However, neither Mal3-chlorin

nor light applied alone caused any toxicity in AsPC1/luc cells. Similar results were

obtained with the other cell lines (AsPC1, BxPC3 and BxPC3/luc; data not shown).

Futhermore, neither Mal3-chlorin nor talaporfin without irradiation induced any

cytotoxicity (Figure 2B and Supplementary Figure S1, respectively). We next examined

the IC50 of talaporfin and Mal3-chlorin at 24 h after PDT. As summarized in Table 1,

PDT with Mal3-chlorin induced cell death with 5-27 times more cytotoxicity to all

cancer cells than PDT with talaporfin. At the same time, in AsPC1/luc cells, we

confirmed that the luciferase luminescence intensities reduced in parallel with cell

viability by luciferase assay (Supplementary Figure S2).

Moreover, the level of apoptosis after PDT with Mal3-chlorin was increased in a

dose-dependent manner compared to after PDT with talaporfin, whereas neither

Mal3-chlorin nor talaporfin alone induced apoptosis (Figure 2C). To further explore

these cytotoxic effects, we then analyzed the levels of intracellular ROS in AsPC1/luc




15

cells using the DCFH-DA assay and found significantly elevated ROS intensities after

PDT with Mal3-chlorin (Figure 2D and 2E). Likewise, the H2O2 assay also showed

dose-dependent increases in ROS generations after PDT with Mal3-chlorin that were

higher than the increases observed after PDT with talaporfin (Figure 2F).

Mal3-chlorin accumulates in xenograft tumors of pancreatic cancer

We examined the ability of Mal3-chlorin to accumulate in xenograft tumors established

by subcutaneously implanting AsPC1/luc pancreatic cancer cells into mice. At 2 weeks

after the cell injections, we first confirmed successful cell implantation by

bioluminescence imaging (Figure 3A). Then, the peritoneal cavities were washed with

saline and imaged under white light (Figure 3B), followed by fluorescence imaging of

the same view at 4 h after i.p. injection of 1.25 μmol/kg Mal3-chlorin. We observed the

characteristic red fluorescence of Mal3-chlorin in the disseminated peritoneal tumors

(Figure 3C). Similarly, following an i.p. injection of 1.25 μmol/kg talaporfin or

Mal3-chlorin, tumor tissue and some organs in each group were excised and observed

under white light and fluorescence imaging (Figure 3D). We then measured the

biodistribution of talaporfin and Mal3-chlorin in xenograft models by spectrophotometry.

Notable accumulation of Mal3-chlorin in the tumor tissue was detected (Figure 3E),




16

while among the organ tissues, Mal3-chlorin tended to accumulate in liver and stomach,

although at a much lower level than those in tumor. The stomach accumulation was

measured at the lower stomach due to the strong autofluorescence of the upper stomach.

PDT with Mal3-chlorin suppressed tumor progression in a mouse model of

disseminated peritoneal pancreatic cancer

To investigate the anti-tumor effects of PDT with Mal3-chlorin on intraperitoneal

dissemination in vivo, each PDT was performed on a pancreatic cancer model

established by i.p. implantation of AsPC1/luc cells into nude mice, as shown in Figure

4A. All animals remained healthy throughout the experimental period, and there was no

significant difference in the mean body weight among the groups at the end of the study

(Supplementary Figure S3). We detected the inhibitory effect of PDT with Mal3-chlorin

on intraperitoneal dissemination with bioluminescence imaging (Figure 4B), although

there was no effect on abdominal skin (Supplementary Figure S4). Numeric evaluations

revealed that PDT with Mal3-chlorin significantly suppressed tumor growth compared

with control treatment (P = 0.036) and tended to suppress it more than PDT with

talaporfin (P = 0.074) (Figure 4C). We also observed that PDT with Mal3-chlorin tended

to inhibit the incidence of ascites formation (Supplementary Table S1) and the mean




17

volume of ascites compared to mice in the control and PDT with talaporfin group (P =

0.066 and P = 0.159, respectively) (Figure 4D).

PDT with Mal3-chlorin induced apoptosis in peritoneal disseminated tumor

Peritoneal tumor tissues from the PDT with Mal3-chlorin group showed some

chromatin-condensed single cells, therefore TUNEL assays were performed and

positivity was found in such dead lesions (Figure 5A). PDT with Mal3-chlorin

significantly increased the TUNEL apoptotic indices compared with control treatment or

PDT with talaporfin (Figure 5B), and additional immunohistochemistry revealed some

TUNEL-positive cells expressing cleaved caspase-3 (Figure 5A). In contrast, cleaved

caspase-3-positive cells were scarcely found in peritoneal tumors of control and PDT

with talaporfin-treated tumors.

Discussion

The poor outcome of pancreatic cancer is due, at least in part, to peritoneal

dissemination (3, 4). In pancreatic cancer patients with peritoneal dissemination, the

lack of effective chemotherapeutic and targeted agents highlight the urgent need for

novel treatment strategies. Against this background, PDT is emerging as a clinically




18

promising locoregional therapy for pancreatic cancer that could also provide aggressive

treatment for peritoneal dissemination in partnership with chemotherapy. Such therapy

could also be useful to control ascites resulting in improved quality of life during

palliative treatments. Supporting this promise, we demonstrated herein a suppressive

effect of PDT with Mal3-chlorin in a mouse model of disseminated peritoneal pancreatic

cancer, based on in vivo luminescent imaging and measurement of ascites volume. We

could not elucidate the median survival time in the scope of this study within the

protocols approved by the Institutional Animal Care and Use Committee. However, we

expect that PDT with Mal3-chlorin could improve survival rates in such experimental

models compared to untreated mice due to the reduced volumes of both peritoneal

tumor and ascites.

At clinical sites, the tumoritropic characteristics of photosensitizers become crucial in

reducing the damage to adjacent normal tissue. There have been many attempts to

develop new photosensitizers that show preferential accumulation within the target

tumor tissue through combinations with various active targeting approaches, such as

conjugation with peptides or antibodies (23-26), incorporation within liposomes (27,

28) and encapsulation within polymeric nanoparticles (29-32). To initiate accumulation

in the target tumor, G-chlorin was initially synthesized by linking glucose to the




19

photosensitizer chlorine based on tumors consuming higher levels of glucose than

normal cells and indeed, G-chlorin showed improved cancer cell targeting compared to

chlorin and other photosensitizers (5, 17, 18). For clinical use, we recently developed

Mal3-chlorin introduced water-solubility. As for the 1O2 generation efficiency and

photocytotoxicity, Mal3-chlorin was proved to have a very high performance

comparable to that of G-chlorin. Additionally, the crucial water-soluble advantage of

Mal3-chlorin allows its injection without the use of toxic organic solvents, with

hydrophilicity similar to that of talaporfin (20).

Talaporfin, a second-generation photosensitizer, shows improved efficacy compared

with first-generation photosensitizers (12). Now, our in vivo results indicate that

Mal3-chlorin-mediated PDT tends to be more effective than talaporfin-mediated PDT at

a low photosensitizer concentration (1.25 μmol/kg). Although we could not find the

effects of PDT with talaporfin, we speculate that talaporfin required over five times

more dose ( > 6.25 μmol/kg) than the dose which was used in the present study,

according to a previous study (33, 34). This indicated that Mal3-chlorin was more

efficiently taken up by pancreatic cancer cells than talaporfin. In support of this,

previous PDT experiments with photosensitizers including talaporfin used irradiation

doses of >100 J/cm2 in carcinoma xenografts models (33, 35, 36), while in this study,




20

PDT with Mal3-chlorin showed antitumor effects in vitro and in vivo at a low irradiation

dose of 13.9 J/cm2. The high cancer cell selectivity of Mal3-chlorin could therefore

substantially reduce the total energy of light irradiation needed in PDT treatment.

Glucose transporter 1 (GLUT1) is believed to maintain basal glucose transport in most

cell types (37, 38) and is predominant in many types of cancer, including pancreatic

cancer (39). We investigated the correlation of GLUT1 to the uptake of Mal3-chlorin

using a pharmacological GLUT1 inhibitor, WZB117 (40), and found that WZB117

inhibited the uptake of Mal3-chlorin (Supplementary Figure S5). The experiment also

hinted at an involvement for GLUT1 in the mechanism by which Mal3-chlorin is taken

up by cancer cells, although what underlies such a mechanisms remains unclear. In

addition, several recent reports showed that mutated KRAS caused higher glucose

uptake and accumulation possibly by upregulation of GLUT1 (41, 42). KRAS mutations

occur in more than 90% of pancreatic cancers, thus G-chlorin and Mal3-chlorin might

produce more effects in pancreatic cancer than in other cancers expressing wild-type

KRAS.

Importantly, Mal3-chlorin might have additional, prospective value as a photodynamic

diagnosis (PDD) reagent to detect and discriminate tumor locations due to its high

cancer cell selectivity. Our in vitro findings revealed dose- and cell number-dependent




21

increases in fluorescence intensities with Mal3-chlorin (Supplementary Figure S6).

However, it is currently too difficult to evaluate the correlation between tumor

fluorescence and either the dose of Mal3-chlorin or tumor size in vivo because of mice

inter-individual differences in microvascular construction and tumor microenvironment.

Further studies are therefore needed to establish the usefulness of Mal3-chlorin in PDD.

It should also be noted that the xenograft peritoneal cancer mouse model used in the

present study shows differences in microenvironment compared to tumors from human

patients, including in the vasculature, stromal cells, and immune cells (43, 44). We

therefore plan to further investigate the effects shown here in a future study using a

spontaneous peritoneal disseminated model.

Collectively, our results indicated that PDT with Mal3-chlorin could effectively

suppress the growth of peritoneal disseminated xenograft mouse models of human

pancreatic cancer cells through apoptosis without observable damage to the adjacent

normal tissue. Further, the newly developed water-soluble Mal3-chlorin had superior

cancer cell selectivity without skin phototoxicity (Supplementary Figure S4). Thus,

Mal3-chlorin stands as a candidate for the next-generation photosensitizers and our

findings may offer new opportunities for the clinical use of PDT in treating peritoneal

dissemination of pancreatic cancer.




22

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA: a cancer journal for

clinicians. 2015;65:5-29.

2. Nakachi K, Furuse J, Ishii H, Suzuki E-i, Yoshino M. Prognostic Factors in

Patients with Gemcitabine-Refractory Pancreatic Cancer. Japanese Journal of Clinical

Oncology. 2007;37:114-20.

3. Abou-Alfa GK, Letourneau R, Harker G, Modiano M, Hurwitz H, Tchekmedyian

NS, et al. Randomized phase III study of exatecan and gemcitabine compared with

gemcitabine alone in untreated advanced pancreatic cancer. J Clin Oncol. 2006;24:4441-7.

4. Berlin JD, Catalano P, Thomas JP, Kugler JW, Haller DG, Benson AB, 3rd. Phase

III study of gemcitabine in combination with fluorouracil versus gemcitabine alone in

patients with advanced pancreatic carcinoma: Eastern Cooperative Oncology Group Trial

E2297. J Clin Oncol. 2002;20:3270-5.

5. Yano S, Hirohara S, Obata M, Hagiya Y, Ogura S-i, Ikeda A, et al. Current states

and future views in photodynamic therapy. Journal of Photochemistry and Photobiology C:

Photochemistry Reviews. 2011;12:46-67.

6. Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nature

reviews Cancer. 2003;3:380-7.

7. Abels C. Targeting of the vascular system of solid tumours by photodynamic

therapy (PDT). Photochem Photobiol Sci. 2004;3:765-71.

8. Allison RR, Moghissi K. Photodynamic Therapy (PDT): PDT Mechanisms. Clin

Endosc. 2013;46:24-9.

9. Li Z, Agharkar P, Chen B. Therapeutic enhancement of vascular-targeted

photodynamic therapy by inhibiting proteasomal function. Cancer Lett. 2013;339:128-34.

10. Juarranz A, Jaen P, Sanz-Rodriguez F, Cuevas J, Gonzalez S. Photodynamic

therapy of cancer. Basic principles and applications. Clinical & translational oncology :

official publication of the Federation of Spanish Oncology Societies and of the National

Cancer Institute of Mexico. 2008;10:148-54.

11. Triesscheijn M, Baas P, Schellens JH, Stewart FA. Photodynamic therapy in

oncology. Oncologist. 2006;11:1034-44.

12. Gomer CJ, Ferrario A. Tissue distribution and photosensitizing properties of

mono-L-aspartyl chlorin e6 in a mouse tumor model. Cancer research. 1990;50:3985-90.

13. Horimatsu T, Muto M, Yoda Y, Yano T, Ezoe Y, Miyamoto S, et al. Tissue damage in

the canine normal esophagus by photoactivation with talaporfin sodium (laserphyrin): a




23

preclinical study. PloS one. 2012;7:e38308.

14. Kato H, Furukawa K, Sato M, Okunaka T, Kusunoki Y, Kawahara M, et al. Phase

II clinical study of photodynamic therapy using mono-<span

class="small">l</span>-aspartyl chlorin e6 and diode laser for early superficial squamous

cell carcinoma of the lung. Lung Cancer.42:103-11.

15. Kujundžić M, Vogl TJ, Stimac D, Rustemović N, Hsi RA, Roh M, et al. A Phase II

safety and effect on time to tumor progression study of intratumoral light infusion

technology using talaporfin sodium in patients with metastatic colorectal cancer. Journal of

Surgical Oncology. 2007;96:518-24.

16. Yano T, Muto M, Minashi K, Iwasaki J, Kojima T, Fuse N, et al. Photodynamic

therapy as salvage treatment for local failure after chemoradiotherapy in patients with

esophageal squamous cell carcinoma: A phase II study. International Journal of Cancer.

2012;131:1228-34.

17. Tanaka M, Kataoka H, Mabuchi M, Sakuma S, Takahashi S, Tujii R, et al.

Anticancer effects of novel photodynamic therapy with glycoconjugated chlorin for gastric

and colon cancer. Anticancer Res. 2011;31:763-9.

18. Tanaka M, Kataoka H, Yano S, Ohi H, Moriwaki K, Akashi H, et al. Antitumor

Effects in Gastrointestinal Stromal Tumors Using Photodynamic Therapy with a Novel

Glucose-Conjugated Chlorin. American Association for Cancer Research. 2014;13:767-75.

19. Warburg O. On the Origin of Cancer Cells. Science (New York, NY).

1956;123:309-14.

20. Narumi A, Tsuji T, Shinohara K, Yamazaki H, Kikuchi M, Kawaguchi S, et al.

Maltotriose-conjugation to a fluorinated chlorin derivative generating a PDT photosensitizer

with improved water-solubility. Org Biomol Chem. 2016;14:3608-13.

21. Suzuki S, Pitchakarn P, Sato S, Shirai T, Takahashi S. Apocynin, an NADPH

oxidase inhibitor, suppresses progression of prostate cancer via Rac1 dephosphorylation.

Experimental and toxicologic pathology : official journal of the Gesellschaft fur

Toxikologische Pathologie. 2013;65:1035-41.

22. Kato A, Naiki-Ito A, Nakazawa T, Hayashi K, Naitoh I, Miyabe K, et al.

Chemopreventive effect of resveratrol and apocynin on pancreatic carcinogenesis via

modulation of nuclear phosphorylated GSK3beta and ERK1/2. Oncotarget. 2015;6:42963-75.

23. Bisland SK, Singh D, Gariepy J. Potentiation of chlorin e6 photodynamic activity in

vitro with peptide-based intracellular vehicles. Bioconjug Chem. 1999;10:982-92.

24. Del Governatore M, Hamblin MR, Shea CR, Rizvi I, Molpus KG, Tanabe KK, et al.

Experimental photoimmunotherapy of hepatic metastases of colorectal cancer with a 17.1A

chlorin(e6) immunoconjugate. Cancer research. 2000;60:4200-5.




24

25. Sharman WM, van Lier JE, Allen CM. Targeted photodynamic therapy via receptor

mediated delivery systems. Adv Drug Deliv Rev. 2004;56:53-76.

26. Hayashi N, Kataoka H, Yano S, Tanaka M, Moriwaki K, Akashi H, et al. A novel

photodynamic therapy targeting cancer cells and tumor-associated macrophages. Mol

Cancer Ther. 2015;14:452-60.

27. Chen B, Pogue BW, Hasan T. Liposomal delivery of photosensitising agents. Expert

Opin Drug Deliv. 2005;2:477-87.

28. Ichikawa K, Hikita T, Maeda N, Yonezawa S, Takeuchi Y, Asai T, et al.

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified

with peptide specific to angiogenic vessels. Biochim Biophys Acta. 2005;1669:69-74.

29. Konan YN, Berton M, Gurny R, Allemann E. Enhanced photodynamic activity of

meso-tetra(4-hydroxyphenyl)porphyrin by incorporation into sub-200 nm nanoparticles. Eur

J Pharm Sci. 2003;18:241-9.

30. Loo C, Lowery A, Halas N, West J, Drezek R. Immunotargeted nanoshells for

integrated cancer imaging and therapy. Nano Lett. 2005;5:709-11.

31. Reddy GR, Bhojani MS, McConville P, Moody J, Moffat BA, Hall DE, et al. Vascular

targeted nanoparticles for imaging and treatment of brain tumors. Clinical cancer research :

an official journal of the American Association for Cancer Research. 2006;12:6677-86.

32. Zhao B, Yin JJ, Bilski PJ, Chignell CF, Roberts JE, He YY. Enhanced

photodynamic efficacy towards melanoma cells by encapsulation of Pc4 in silica

nanoparticles. Toxicol Appl Pharmacol. 2009;241:163-72.

33. Ohashi S, Kikuchi O, Tsurumaki M, Nakai Y, Kasai H, Horimatsu T, et al.

Preclinical validation of talaporfin sodium-mediated photodynamic therapy for esophageal

squamous cell carcinoma. PloS one. 2014;9:e103126.

34. Kishi K, Yano M, Inoue M, Miyashiro I, Motoori M, Tanaka K, et al.

Talaporfin-mediated photodynamic therapy for peritoneal metastasis of gastric cancer in an

in vivo mouse model: drug distribution and efficacy studies. Int J Oncol. 2010;36:313-20.

35. Peng Q, Warloe T, Moan J, Godal A, Apricena F, Giercksky KE, et al. Antitumor

effect of 5-aminolevulinic acid-mediated photodynamic therapy can be enhanced by the use

of a low dose of photofrin in human tumor xenografts. Cancer research. 2001;61:5824-32.

36. Lim YC, Yoo JO, Park D, Kang G, Hwang BM, Kim YM, et al. Antitumor effect of

photodynamic therapy with chlorin-based photosensitizer DH-II-24 in colorectal carcinoma.

Cancer science. 2009;100:2431-6.

37. Zhao FQ, Keating AF. Functional properties and genomics of glucose transporters.

Curr Genomics. 2007;8:113-28.

38. Young CD, Lewis AS, Rudolph MC, Ruehle MD, Jackman MR, Yun UJ, et al.




25

Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary

tumor cell growth in vitro and in vivo. PloS one. 2011;6:e23205.

39. Reske SN, Grillenberger KG, Glatting G, Port M, Hildebrandt M, Gansauge F, et al.

Overexpression of glucose transporter 1 and increased FDG uptake in pancreatic carcinoma.

Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

1997;38:1344-8.

40. Wood TE, Dalili S, Simpson CD, Hurren R, Mao X, Saiz FS, et al. A novel inhibitor

of glucose uptake sensitizes cells to FAS-induced cell death. Mol Cancer Ther.

2008;7:3546-55.

41. Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H, et al. Glucose

deprivation contributes to the development of KRAS pathway mutations in tumor cells.

Science (New York, NY). 2009;325:1555-9.

42. Iwamoto M, Kawada K, Nakamoto Y, Itatani Y, Inamoto S, Toda K, et al.

Regulation of 18F-FDG accumulation in colorectal cancer cells with mutated KRAS. Journal

of nuclear medicine : official publication, Society of Nuclear Medicine. 2014;55:2038-44.

43. Frese KK, Tuveson DA. Maximizing mouse cancer models. Nature reviews Cancer.

2007;7:645-58.

44. Cabral H, Murakami M, Hojo H, Terada Y, Kano MR, Chung UI, et al. Targeted

therapy of spontaneous murine pancreatic tumors by polymeric micelles prolongs survival

and prevents peritoneal metastasis. Proc Natl Acad Sci U S A. 2013;110:11397-402.

Table Table 1. The IC50 for pancreatic cancer with PDT using talaporfin and Mal3-chlorin

IC50 (μM) AsPC1 AsPC1/luc BxPC3 BxPC3/luc

Talaporfin 16.59 12.02 11.92 11.23 Mal3-chlorin 1.51 0.45 2.24 0.66 Talaporfin; Talaporfin sodium, Mal3-chlorin; maltotriose-conjugated chlorin, IC50; the half maximal (50%) inhibitory concentration

Figure Legends

Figure 1: Chemical structure and uptake of Mal3-chlorin. A. Chemical structure of

talaporfin. B. Synthesis of Mal3-chlorin by conjugation of four maltotriose molecules to




26

a chlorin derivative. C. Uptake of the photosensitizers in pancreatic cancer cells

(AsPC1/luc) was estimated by flow cytometric analysis.

Figure 2: Cytotoxic effects of PDT with Mal3-chlorin through apoptosis and ROS

generation. A. Representative microscopic images of AsPC1/luc cells treated with (24

h after PDT) or without Mal3-chlorin-mediated PDT (original magnification, × 200). B.

Cell viability in AsPC1/luc and BxPC3/luc cells treated by Mal3-chlorin with or without

irradiation. C. Caspase 3/7 activity in AsPC1/luc cells after PDT. Values are means ±

SD, n = 4. D. and E. ROS production photos (C) and data (D) were detected by

DCFH-DA with (1 h after PDT) or without Mal3-chlorin-mediated PDT. Data are means

± SD values from 3 independent experiments. F. H2O2 concentrations in AsPC1/luc cells

after PDT. Values are means ± SD, n = 4 in each, *P < 0.05, **P < 0.01 and ***P <

0.001 compared to non-treated cells.

Figure 3: Accumulation of Mal3-chlorin in mice disseminated peritoneal tumors.

A-C. Representative disseminated peritoneal tumors in the mice 2 weeks after

AsPC1/luc cell implantation. A: Bioluminescence imaging with in vivo imaging system,

B: White light image, C: Fluorescence image. Arrows indicate disseminated peritoneal




27

tumors and red fluorescence aligns with the peritoneal tumor. D. Images of excised

tumor tissue and some organs in each group (upper panel: white light image, lower

panel: fluorescence image). E. The fluorescence intensity ratio in tumors and some

organs of talaporfin- and Mal3-chlorin-mediated PDT groups was calculated and shown

relative to that in control mice.

Figure 4: Treatment effects of PDT with Mal3-chlorin in a mouse model of

disseminated peritoneal pancreatic cancer. A. The treatment regimen is shown.

Bioluminescence images were obtained at day 0, 7, and 14. B. Representative

bioluminescence imaging of AsPC1/luc peritoneal tumors at day 14 in control mice (left

panel), Talaporfin-mediated PDT mice (middle panel) and Mal3-chlorin-mediated PDT

mice (right panel). C. Quantitative analysis of bioluminescence dynamics in each group

(n = 7 per group). *P < 0.05. D. Volumes of ascites in each group of mice.

Figure 5: Immunohistochemistry and TUNEL indices of peritoneal tumors. A.

Representative immunohistochemical staining for TUNEL and cleaved caspase 3 in

peritoneal tumor tissues of each group. Bars = 100 μm. B. TUNEL indices in peritoneal

tumors form each group (n = 3 in each, *P < 0.05 and **P < 0.01).




Published OnlineFirst March 14, 2017.Mol Cancer Ther Akihisa Kato, Hiromi Kataoka, Shigenobu Yano, et al. dissemination of pancreatic cancerantitumor effects of photodynamic therapy in peritoneal Maltotriose conjugation to a chlorin derivative enhances the

Updated version

10.1158/1535-7163.MCT-16-0670doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2017/03/14/1535-7163.MCT-16-0670.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2017/03/14/1535-7163.MCT-16-0670To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-16-0670

http://mct.aacrjournals.org/content/suppl/2017/03/14/1535-7163.MCT-16-0670.DC1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2017/03/14/1535-7163.MCT-16-0670


akihisa kato1, hiromi kataoka1*, shigenobu yano2, kazuki ... · 3/14/2017 · translational...

Documents