Cancer Research Cancer Epigenetics Genetics and Biology of Brain Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogata, T.
Right arrow Articles by Matsuura, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogata, T.
Right arrow Articles by Matsuura, N.
[Cancer Research 65, 113-120, January 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Particle Irradiation Suppresses Metastatic Potential of Cancer Cells

Toshiyuki Ogata1,3, Teruki Teshima1, Kazufumi Kagawa3, Yoshio Hishikawa3, Yutaka Takahashi1, Atsuko Kawaguchi1, Yuko Suzumoto1, Kumie Nojima4, Yoshiya Furusawa5 and Nariaki Matsuura1

Departments of 1 Medical Physics & Engineering and 2 Molecular Pathology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 3 Department of Radiology, Hyogo Ion Beam Medical Center, Ibo-gun, Hyogo, Japan; and Laboratories of 4 International Space Radiation; and 5 Heavy-ion Radiobiology for Therapy, National Institute of Radiological Sciences, Inage-ku, Chiba, Japan

Requests for reprints: Teruki Teshima, Departments of Medical Physics & Engineering, Osaka University Graduate School of Medicine, 1-7 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 166-879-2570; Fax: 166-879-2570; E-mail: teshima{at}sahs.med.osaka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Particle radiotherapy such as proton and carbon ion has been producing promising clinical results worldwide. The purpose of this study was to compare metastatic capabilities of malignant tumor cells after irradiation with photon, proton, and carbon ion beams to clarify their ion beam–specific biological effects. We examined the biological properties of highly aggressive HT1080 human fibrosarcoma cells to assess their metastatic processes in terms of cell adhesion capability to extracellular matrix, expression of integrins, cell migration, cell invasive capability, and matrix metalloproteinase-2 activity in vitro. We then assessed the metastatic capabilities of LM8 mouse osteosarcoma irradiated with carbon ion or photon beam in the syngeneic mice. Both proton and carbon ion irradiation decreased cell migration and invasion in a dose-dependent manner and strongly inhibited matrix metalloproteinase-2 activity. On the other hand, lower X-ray irradiation promoted cell migration and invasion concomitant with up-regulation of {alpha}Vß3 integrin. For cancer cells treated with carbon ion irradiation, the number of pulmonary metastasis was decreased significantly in vivo. These findings suggest that particle irradiation suppresses metastatic potential even at lower dose, whereas photon irradiation promotes cell migration and invasive capabilities at lower dose level, and provide preclinical evidence that ion beam radiotherapy may be superior to conventional photon beam therapy in possible preventive effects on metastases of irradiated malignant tumor cells.

Key Words: proton • carbon ion • X-ray • integrin • MMP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Metastasis, the biggest threat to survival for patients with solid tumors, is the spread of tumor cells from the original growth to the other sites in the body. Metastatic processes of malignant tumor cells generally consist of (i) detachment of cells from the primary tumor, (ii) migration to extracellular matrices, (iii) degradation of basement membrane, (iv) invasion into blood vessels, (v) circulation in blood flow, (vi) escape to extravascular matrices, and (vii) implantation to target organs. These processes are based upon a number of biological characteristics associated with various molecular changes involving proteinases, adhesion molecules, and cell motility factors.

The integrin family of adhesion molecules is extracellular matrix receptors consisting of {alpha} and ß chains that form various heterodimers with distinct cellular and adhesive characteristics. Integrin-mediated adhesion to extracellular matrix triggers intracellular signaling pathways to modulate cell proliferation, shape, migration, invasion, and survival (1, 2). The ß1 integrin subfamily consists of a receptor subunit associated with several {alpha} subunits resulting in a broad spectrum of receptors for a variety of potential ligands (3, 4). The vitronectin receptor, {alpha}Vß3 integrin, also seems to be associated with increased invasiveness (5, 6).

Matrix metalloproteinases (MMP) constitute a family of Zn2+-dependent enzymes essential for extracellular matrix turnover under normal and pathologic conditions. Especially MMP-2 can degrade type IV collagen, one of the major components of the basement membrane, resulting in the promotion of tumor invasion and metastasis (7). One of the mechanisms of this process is that MMP-2 directly binds to {alpha}Vß3 integrin and thus localizes in a proteolytically active form on the surface of invasive cells (8).

In the clinic, ionizing radiation has been established as a highly effective modality used in the local control of tumor growth. However, several authors have reported that photon beam irradiation enhanced metastatic processes of malignant tumor cells at sublethal dose (9–13). New types of radiation sources, particle beams such as proton and carbon ion, may be expected to be a new modality of cancer treatment. Particle therapy has the advantage, in theory, over conventional photon beam that the tumor can be targeted with extreme precision, without damage to normal surrounding tissue, either superficial or deep, thereby allowing for an extraordinary escalation of dosage to the tumor. Carbon ion with high linear energy transfer has been shown more effective than photon and proton for cell-killing effect (14–16). Only a few studies have been conducted of the effects of particle beams on functioning of cells with metastatic potential. Our group was the first to report that carbon beam irradiation inhibited in vitro angiogenesis even at sublethal dose (17).

We show metastatic potential after irradiation with photon, proton, and carbon ion beams to elucidate particle-specific biological effects. Here, we report that particle irradiation suppresses metastatic potential, whereas photon irradiation promoted cell migration and invasive capabilities at lower dose level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Reagents. Highly aggressive HT1080 human fibrosarcoma (American Type Culture Collection, Rockville, MD) and LM8 mouse osteosarcoma (18), a highly metastatic Dunn cell subline which was kindly given by Dr. Yoshikawa (Osaka University, Osaka, Japan), were maintained in DMEM medium (Nihonseiyaku, Tokyo, Japan) with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 10% CO2 and 90% air. The MMP inhibitor GM6001 was purchased from Chemicon (Temecula, CA).

Irradiation. Cell irradiation with 190 MeV/nucleon proton beams was done at the Hyogo Ion Beam Medical Center in Japan. Cells were irradiated at the center of Bragg peaks modulated to 6-cm widths. The irradiation system and biophysical characteristics of proton beams have been detailed elsewhere (19).

For carbon ion irradiation, cells were treated with 290 MeV/nucleon carbon ion beams at 6-cm spread-out Bragg peak center from the Heavy Ion Medical Accelerator in Chiba at the National Institute of Radiological Sciences in Japan. The irradiation system for carbon ion at Heavy Ion Medical Accelerator in Chiba and the physical characteristics of the beam have been described elsewhere (20, 21).

For photon irradiation, 4 MV X-ray from the linear accelerator at Osaka University Graduate School of Medicine was used with a delivered dose rate of ~1.8 Gy/min.

Colony Formation Assay. Survival curves were obtained by means of standard colony formation assay. Irradiated cells were plated onto triplicate 60-mm-diameter plastic dishes aiming for 80 to 100 colonies per dish. After 10 to 12 days of incubation, colonies were fixed with 10% formalin and stained with crystal violet. Colonies with >50 cells were scored as a surviving colony.

Cell Adhesion Assay. Plastic plates (96 wells) were coated with 10 µg/mL of collagen, laminin, fibronectin, and vitronectin (IWAKI, Chiba, Japan) in PBS (Invitrogen) for 2 hours at 37°C and then treated with 3% bovine serum albumin for 1 hour at 37°C, or were coated with only bovine serum albumin for negative control. The cells (2 x 105 cells/mL) in serum-free DMEM containing 0.1% bovine serum albumin were then added and incubated for 2 hours at 37°C. After removal of the medium, a 0.04% crystal violet solution was added and incubation was conducted for 10 minutes at room temperature. The wells were washed thrice with PBS and 20 µL of Triton X-100 were added for permeabilization. Finally, distilled water was then added for a total quantity of 100 µL, and the number of adherent cells was assessed with a microplate reader (measurement wavelength = 550 nm and reference wavelength = 630 nm).

Flow Cytometry. For {alpha}Vß3 and ß1 integrin analysis, cells in DMEM supplemented with 1% fetal bovine serum and 0.03% sodium azide were incubated with amonoclonal antibody against mouse monoclonal antibody {alpha}Vß3 and ß1 (Chemicon), for 30 minutes at 4°C. After washing with DMEM as described above, the cells were incubated with FITC-conjugated mouse IgG (DAKO, Copenhagen, Denmark) for 30 minutes at 4°C. After washing, cells were resuspended with the same medium and analyzed using a FACSCalibur (Beckton Dickson, Heidelberg, Germany) with CellQuest software (Beckton Dickson). Finally, cell surface fluorescence for individual integrin receptors was obtained.

Chemotaxis Assay. Chemotaxis was assessed with a 48-microwell chemotaxis chamber (Neuro Probe, Gaithersburg, MD) that was set a polycarbonate filter of 8-µm pores coated with 10 µg/mL fibronectin. The cells were trypsinized, resuspended in 0.1% bovine serum albumin and adjusted to a final concentration of 1 x 106 cells/mL. The cells (5 x 104) were added to the upper well, which was placed into a lower well containing medium with 10% fetal bovine serum as a chemoattractant. After 3 hours of incubation at 37°C, cells remaining on the upper membrane surface were removed with a cotton swab. The cells that had migrated to the bottom of the filter were fixed with formalin and stained with hematoxylin. Cell migration was quantitated by counting the number of stained nuclei in four random fields at 20x magnification with a microscope.

Matrigel Invasion Assay. Invasion of cancer cells was assessed by measuring the invasion of cells through transwell inserts with 8-µm pores coated with Matrigel (Becton Dickinson). Irradiated cells were trypsinized, washed twice with DMEM supplement with 0.1% bovine serum albumin, and 200 µL of cell suspension (5 x 105 cells/mL) per condition were added to the upper well. DMEM supplement with 10% fetal bovine serum (700 µL) as a chemoattractant was added to the lower well. The number of cells that had invaded to the lower surface of the Matrigel-coated membrane was counted in four random fields under a microscope.

Gelatin Zymography. MMP-2 activity was analyzed as detailed elsewhere (22). After irradiation, cells were washed twice with PBS and incubated with serum-free DMEM for 24 hours. After the medium had been centrifuged to remove corpuscular material, supernatant was collected, frozen in liquid nitrogen, and stored at –80°C. Samples were mixed with SDS sample buffer without heating or reduction and applied to 8% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, gels were renatured by soaking for 45 minutes at room temperature in 2.5% Triton X-100 with gentle agitation and then incubated for 12 hours at 37°C in buffer containing 5 mmol/L CaCl2 and 1 µmol/L ZnCl2. Gelatinolytic activity made the bright bands visible at Mr 72,000 for the pro form and Mr 62,000 for the active form ofMMP-2.

Animal and Tumor Model. LM8 cells were irradiated with 290 MeV/nucleon carbon ion beams or 4 MV X-ray (proton irradiation was not done because of restricted irradiation time). Cells were harvested by treatment with trypsin-EDTA (Invitrogen), washed twice with serum-free DMEM, and suspended in serum-free DMEM. Irradiated LM8 mouse osteosarcoma cells, 105 cells in 0.05 mL, were injected s.c. into the hind limbs or inoculated into tail vein of 8- to 10-week-old female specific pathogen-free C3H/HeJ mice (Charles River, Yokohama, Japan). In s.c. tumor, tumor volume (mm3) was measured with calipers and calculated according to the formula: 1/2 x length x width2. Mice injected s.c. or i.v. were euthanized 15 or 30 days after injection. Lung tumor formation was observed under a dissecting stereomicroscope, and the number of lung tumors was counted. These experiments were repeated twice.

Statistics. The results were expressed as mean values with SDs of at least three independent experiments, except indicated elsewhere. The statistical significance was tested by means of Student's t test or ANOVA where appropriate. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Survival Curves of Cancer Cells. To require biologically equivalent doses for each radiation quality, we first examined clonogenic survival using the colony formation assay. For HT1080 cells, the relative biological effectiveness values, calculated by the D10 relative to X-ray, were to be 1.1 for proton irradiation and 1.9 for carbon irradiation (Fig. 1)). The corresponding relative biological effectiveness measured by the D10 relative to X-rays for the LM8 cells was 2.3 for carbon irradiation (Fig. 1). Therefore, in subsequent assays, we applied the physical doses of carbon ion or proton to half or the same physical doses for X-ray.



View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1 Clonogenic survival curves after photon, proton, or carbon beam irradiation for cancer cells. Surviving fractions against physical doses were plotted and fitted to surviving curves using the following linear-quadratic model: SF = exp(–{alpha}D–ßD2), where SF is the surviving fraction and D is the physical dose. Survival curve for proton irradiation was examined only in HT1080 cells.

 
The Expression Levels of Integrin and Adhesion to Extracellular Matrix. Cell adhesion assays were done to assess tumor cell adhesion capabilities to extracellular matrix proteins (collagen, fibronectin, laminin, and vitronectin). The adhesion capability to fibronectin, laminin, and vitronectin of cells irradiated with 8 Gy for X-ray or proton had significantly increased 24 hours after irradiation (Fig. 2Aand B). Irradiation with 4 Gy for carbon at 24 hours after irradiation showed a significantly higher attachment to fibronectin compared to the nontreatment controls (Fig. 2C. On the other hand, no significant changes were observed 48 hours after X-ray, proton, and carbon ion irradiation (Fig. 2A-C. Mean fluorescence intensity (% control) of integrins that plays crucial roles in cell adhesion to extracellular matrices, cell migration, and invasion was analyzed by flow cytometry analyses. The expression levels of ß1 integrin did not show significant differences for X-ray, proton, and carbon ion irradiation at 24 hours after irradiation (Table 1)). After delivery of more than 4 Gy of each type of irradiation, the amount of ß1 integrin was increased, although not significantly. The expression levels of {alpha}Vß3 integrin were not changed by either proton or carbon ion irradiation (Table 1). However, for cells irradiated with 0.5 Gy of X-ray, the amount of {alpha}Vß3 integrin was significantly increased compared with untreated controls (Table 1).



View larger version (45K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2 Effects of irradiation on adhesion of cancer cells to collagen (COL), laminin (LM), fibronectin (FN), or vitronectin (VN). A, cells were untreated or irradiated with photon beams. White bar, untreated control cells; dark gray bar, cells irradiated at 0.5 Gy; light gray bar, cells irradiated at 2 Gy; and black bar, cells irradiated at 8 Gy. B, cells were untreated or irradiated with proton beam. White bar, untreated control cells; dark gray bar, cells irradiated with 0.5 Gy; light gray bar, cells irradiated with 2 Gy; and black bar, cells irradiated with 8 Gy. C, cells were untreated or irradiated with carbon ion beam. White bar, untreated control cells; dark gray bar, cells irradiated at 0.2 Gy; light gray bar, cells irradiated at 1 Gy; and black bar, cells irradiated at 4 Gy. Columns, mean; bars, ± SD. *, P < 0.05 (Student's t test, compared with untreated cell).

 

View this table:
[in this window]
[in a new window]

  		Table 1. Effects of irradiation on the expression levels of ß1 and {alpha}Vß3 integrin in cancer cells by flow cytometric analysis

 
Effects of Irradiation on Cell Migration and Invasion. Cell migration and invasion are fundamental components of tumor cell metastasis. To assess the effect of photon, proton, and carbon ion beams on cell motility, we examined the migration of malignant cells 24 and 48 hours after irradiation using chemotaxis assay. For proton as well as carbon ion irradiation, suppression of migration of irradiated cells became apparent at 24 and 48 hours after irradiation in a dose-dependent manner (Fig. 3A and B). On the other hand, an increase in migration was observed by lower dose (0.5 Gy) of X-ray irradiation at 48 hours after irradiation (Fig. 3B. At similar cell killing doses, proton or carbon particle irradiation, compared to X-rays, inhibited the migration (for carbon irradiation except 24 hours after 2 Gy, P < 0.05; At 24 hours after 8 Gy or at 48 hours after proton irradiation, P < 0.05).



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Effects of irradiation on cell migration of HT1080 cells. HT1080 cells were exposed to X-ray ({square}), proton ({bullet}), or carbon ({circ}). Vertical axis, no. migrated cells (%control); horizontal axis, physical dose. Points, mean; bars, ± SD. *, P < 0.05; **, P < 0.01 (ANOVA, compared particle beams with X-ray at similar killing doses).

 
We next focused on changes in the invasive capability of cancer cells after irradiation using the Matrigel invasion assay. Proton as well as carbon ion beam irradiation significantly reduced the invasion capabilities of irradiated cells (Fig. 4A and B). X-ray irradiation promoted cell invasion even at the dose levels below 2Gy (Fig. 4)A and B). Remarkably, invasive potentials of malignant cells were significantly increased by about 2-fold at 24 hours after X-ray irradiation at dose of 2 Gy (Fig. 4A. For cells irradiated with 8 Gy at 48 hours after X-ray irradiation, invasion capabilities were decreased as compared with untreated controls (Fig. 4B. Proton or carbon ion irradiation at comparable cell-killing doses resulted in significantly diminished invasion capabilities 24 or 48 hours after irradiation compared to that resulting from X-ray irradiation (P<0.01).



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Effects of irradiation on cell invasion of HT1080 cells. HT1080 cells were irradiated with X-ray ({square}), proton ({bullet}), or carbon ({circ}). Vertical axis, no. invaded cells (% control); horizontal axis, physical dose. Points, mean; bars, ± SD. *, P < 0.01 (ANOVA, compared particle beams with X-ray at comparable cell killing doses).

 
Effects of Irradiation on MMP-2 Activity. The process of tumor cell invasion and metastasis requires the degradation of connective tissue associated with vascular basement membranes and interstitial connective tissue. Therefore, MMP-2 activity required for tumor invasion was examined by gelatin zymography. Gelatin zymography revealed that proton and carbon ion irradiation strongly inhibited MMP-2 activity of cancer cells in a dose-dependent manner (Fig. 5A). For cells irradiated with X-ray, MMP-2 activity were not changed compared with untreated controls (Fig. 5A).



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Effects of irradiation on MMP-2 activity of cancer cells. Supernatants from untreated or irradiated cells were collected 24 hours after irradiation and analyzed by zymography for pro form (72 kDa) or active form of MMP-2 (62 kDa). Untreated samples in addition to concanavalin A (conA) were used for positive control. A, cells were untreated or irradiated with X-ray, proton, or carbon ion. B, cells were untreated or irradiated with X-ray in the presence or absence of the MMP inhibitor. MMP inhibitor (25 µmol/L) was added in condition medium just after irradiation.

 
The Influence of MMP Inhibitor on Photon-Enhanced Cell Invasion. Particle irradiation inhibited invasion capability due to an association with inhibition of MMP-2 activity. Therefore, we investigated whether the MMP inhibitor could prevent photon-induced increase in invasive potential. The addition of the MMP inhibitor GM6001 to the upper well resulted in a marked reduction of photon-induced invasion (Table 2). Furthermore, gelatin zymography showed that administration of GM6001 at concentration of 25 µmol/L reduced the active form of MMP-2 (Fig. 5B.


View this table:
[in this window]
[in a new window]

  		Table 2. The influence of MMP inhibitor on photon-enhanced cell invasion

 
The Effect of Irradiated Cells on Metastatic Capabilities In vivo. To investigate whether irradiated cells affect metastastic capabilities in vivo, LM8 osteosarcoma cells irradiated with X-ray or carbon ion were injected s.c. into right thighs or i.v. into tail vein of mice. In s.c. tumor, the tumor volumes obtained from irradiated cells were decreased as compared with those of untreated cells (Fig. 6A and B). There was no difference in the volume of tumors from cells treated with X-ray at doses between 2 and 10 Gy (Fig. 6A. However, for cells irradiated with carbon ion, tumor volume was decreased dose dependently (Fig. 6B). Pulmonary metastases of cancer cells irradiated with X-ray did not changed in comparison with those of untreated controls (Fig. 6C), whereas treatment with carbon ion reduced the number of lung metastases in a dose-dependent manner (Fig. 6D).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Metastatic capabilities of cancer cells irradiated with photon or carbon beam in vivo. Growth curves in subcutaneous tumors of LM8 osteosarcoma cells unirradiated, irradiated with X-ray (A), or carbon ion (B). The number of lung metastases treated with X-ray (C), or carbon ion (D) in subcutaneous tumor. Representative of lung metastasis for mice injected intravenously (E). Therapeutic effects of cells irradiated with X-ray (F) or carbon ion (G) on experimental pulmonary metastasis from mice inoculated intravenously. Columns, mean; bars, ± SD. *, P < 0.05 and **, P < 0.01 (Student's t test versus the untreated group).

 
For mice inoculated i.v., X-ray irradiation resulted in a 1.2-fold increase in the number of metastatic lung nodules in mice as compared to mice injected with untreated cells (Fig. 6E and F). However, a significant suppression of lung metastases was observed in cells irradiated with carbon ion (Fig. 6E and G).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Metastasis brings about the greatest threat to the survival and quality of life for cancer patients. The ultimate goal of cancer therapy is to treat the primary tumor and any underlying metastases. Particle radiotherapy such as proton and carbon ion has established its efficacy by demonstrating superb results (23–28). The advantage of particle beams over photon is superior distribution of radiation dose due to the physical characteristics, which makes it possible to spare normal tissues close to the target. However, the effects of particle beams on metastatic potential of cancer cells are not yet well understood. We hypothesized that particle beams might inhibit metastatic potential for ion beam-specific biological effects and first focused on the in vitro models including adhesion, migration, invasion, and the expression level or activity of molecules related to metastasis such as {alpha}Vß3, ß1 integrin, and MMP-2.

Various factors are related to metastatic potentials. Changes in integrin expression level are likely to affect cell adhesion closely linked cell functions. X-ray, proton, and carbon ion irradiation of more than 4 Gy was seen to increase significantly cell adhesion capability to extracellular matrix significantly. Cordes et al. (29, 30) showed that radiation-induced increase in adhesion capacity could be modulated by radiation-induced increase in ß1 integrin expression. However, our findings showed that the expression levels of ß1 integrin were increased (≥4 Gy irradiation) but did not show significant differences among X-ray, proton, and carbon ion irradiation. The reason for these discrepant results may be that the use of flow cytometry does not enable us to detect ß1 integrin affinity but only the expression level of ß1 integrin. Integrin affinity for extracellular matrix can be regulated by intracellular signals such as the Ras–, R-Ras– and Rap1-GTPases (31, 32). ß1 Integrin transduces biochemical signals from the extracellular environment, especially with respect to cell survival. It seems likely that radiation (≥4 Gy) may activate ß1 integrin affinity and thus leading radiation-induced (≥4 Gy) increase in adhesion capacity due to cell survival.

Cell migration and invasion are fundamental components of tumor cell metastasis. Wild-Bode et al. (9) reported that sublethal dose of X-ray irradiation induced the expression levels of the {alpha}Vß3 integrin of glioblastoma and led to enhancement of cell migration. We confirmed that X-ray irradiation promotes cell migration capabilities concomitant with the up-regulation of {alpha}Vß3 integrin at lower dose level. However, our study showed that both proton and carbon ion irradiation significantly decreased cell migration and invasion capabilities in a dose-dependent manner. Many studies have shown that MMP-2 plays a critical role in tumor invasion. There have been many reports on the enhancement of MMP-2 activity by X-ray irradiation (9, 12, 33, 34) . One of the mechanisms of this enhancement is that the activation of wild-type p53 by photon irradiation and the resulting increase in MMP-2, which can promote radiation-induced metastasis. Bian and Sun (35) reported that the 5' flanking region of the MMP-2 gene contains a perfect p53 binding sequence and that the binding of wild-type p53, but not mutant p53, to this site up-regulates MMP-2 gene expressions. In a previous study, for HT1080 cells expressed wild-type p53, {gamma}-ray irradiation with doses from 4 to 15 Gy up-regulated this expression (36). Our study showed that MMP-2 was strongly inhibited by carbon ion and proton irradiation. Therefore, invasion capabilities of irradiated cells were significantly suppressed by particle beams. Furthermore, we confirmed that MMP inhibitor blocked the photon-enhanced invasion of cancer cells. Our results concur with Wild-Bode's report that administration of o-phenantroline that is one of the MMP inhibitors significantly inhibited photon-induced invasiveness. Asakawa et al. (37) showed that p53-dependent radiation-induced growth inhibition of SAS tongue carcinoma cells transplanted into nude mice was observed following X-ray irradiation but not carbon ion irradiation. Our finding suggests that particle beam irradiation is not affected by p53 status.

The phenomena underlying the suppression of metastatic capability by particle irradiation in vitro were studied further by investigating metastatic potentials of cancer cells irradiated with carbon ion or photon beams in vivo. For mice inoculated s.c. or i.v., treatment with carbon ion reduced the number of lung metastases in a dose-dependent manner as compared with untreated controls. For several experimental tumors, inadequate X-ray radiation resulted in an increase in metastasis (38). One possible explanation for this increase is that radiation-induced DNA changes increase the metastatic potential of cancer cells (39). Our data suggest that carbon ion irradiation induced DNA changes which suppressed the metastatic capabilities of tumor cells, leading to suppression of pulmonary metastases in vivo. This may have been caused by carbon ion irradiation producing a higher proportion of double-strand DNA breaks than does X-ray irradiation.

In this study, the focus was to elucidate the effects of particle beam on metastatic potential of cancer cells. However, little is known about the basic radiobiological effects of particle beam except for the end point of cell survival, especially about the effects on metastatic capabilities. To date, a few groups have reported on the effects of particle beams on cell functions associated with metastatic capabilities. Our group showed that carbon ion irradiation inhibited MMP-2 activity and down-regulated {alpha}Vß3 integrin, thus leading to inhibition of in vitro angiogenesis (17). Ando et al. (40) reported that the induction by carbon ion irradiation of vascular endothelial growth factor that plays an important role in tumor growth and metastasis. However, lung carcinoma cells irradiated with carbon ion induced vascular endothelial growth factor mRNA expression and increased protein levels dose dependently. Particle therapy still has much room to be studied for optimum use in clinical oncology compared with conventional photon beam treatment. Further intensive studies are also necessary to elucidate the relevant molecular mechanisms specifically related to particle irradiation. In future experiments, other carcinoma cell lines will be examined to confirm that this phenomenon is not specific to one cell line.

The phenomena we observed in this study have two significant impacts on the clinic. First, with advent of recent high precise modality such as intensive modulated radiation therapy, radiation oncologists have been focusing on making the radiation field as small as possible to the clinical target volume. There may be a risk that excellent local controls can be hampered by later increase of distant metastasis. Then, we need individualized radiation field based on such biological behavior of each cancer cell. Second, particles such as proton and carbon may have totally different mechanism of action on cell migration and invasion, because these functions were significantly inhibited even at lower doses of particle. These significant differences in cell functions may be caused by differences in biological mechanisms between particle and electromagnetic wave but cell-killing effect concerning cell survival evaluated with colony formation assay of proton are similar to that of photon.

Photon radiation therapy should be asked with some caution. Lower photon irradiation promotes cell migration and invasive capabilities. However, metastatic capabilities of cancer cells irradiated with 8 Gy of photon beams did not changed in comparison with those of untreated controls. The clinical implications reported by Wild-Bode et al. (9) are that alterations in the fractionation of radiotherapy for human glioblastoma multiform may need to be considered and that inhibitors of migration and invasion may prevent irradiation-induced dissemination of glioma cells from the target volume of irradiation when given during radiotherapy. In addition to these implications, we suggest that not only dose escalation that can eradicate tumors is needed but also examination of the individual radiation field margins, by considering cell migration and identifying microscopic diseases by means of molecular imaging is needed.

To summarize, our study found that particle irradiation de-creased cell migration and invasion in a dose-dependent manner and strongly inhibited MMP-2 activity in vitro. In vivo, treatment with carbon ion reduced the number of lung metastases in a dose-dependent manner. On the other hand, lower X-ray irradiation facilitated cell migration and invasion concomitant with up-regulation of {alpha}Vß3 integrin in vitro.

In conclusion, these data suggests that particle irradiation suppresses metastatic potential even at lower dose whereas photon irradiation promotes cell migration and invasive capabilities at lower dose level. These findings provide preclinical rationales that particle radiotherapy may be superior to conventional photon beam therapy in possible preventive effects on metastases of irradiated malignant tumor cells.


   Acknowledgments
 
Grant support: Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 13670936 and 16390338, Ministry of Health, Labor and Welfare in Japan Grant-in-Aid for Cancer Research 14-6, and a research project with heavy ions at National Institute of Radiological Science-Heavy Ion Accelerator in Chiba (12B621 and 13B132).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sakae Yamamoto, Ayako Madachi, Ryuji Ikeda, Masaki Tanabe, EriAdachi, Kouki Okita, Noriko Ohnishi, and Kimiko Sameshima at the Molecular Pathology Laboratory and Ayaka Kihara at the Radiation Oncology Laboratory for excellent technical support; Dr. Mitsuyuki Abe at Hyogo Ion Beam Medical Center for critical reading of the article and helpful discussions; and the cooperation of all staffers at Hyogo Ion Beam Medical Center in performing proton irradiation.

Received 3/23/04. Revised 10/ 7/04. Accepted 10/31/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Giancotti FG, Ruoslahti E. Integrin signaling. Science 1999;285:1028–32.[Abstract/Free Full Text]
  2. Danen EH, Yamada KM. Fibronectin, integrins, and growth control. J Cell Physiol 2001;189:1–13.[CrossRef][Medline]
  3. Saga S, Chen WT, Yamada KM. Enhanced fibronectin receptor expression in Rous sarcoma virus-induced tumors. Cancer Res 1988;48:5510–3.[Abstract/Free Full Text]
  4. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11–25.[CrossRef][Medline]
  5. Ruoslahti E. The Walter Herbert Lecture. Control of cell motility and tumour invasion by extracellular matrix interactions. Br J Cancer 1992;66:239–42.[Medline]
  6. Felding-Habermann B, Cheresh DA. Vitronectin and its receptors. Curr Opin Cell Biol 1993;5:864–8.[CrossRef][Medline]
  7. Deryugina EI, Luo GX, Reisfeld RA, Bourdon MA, Strongin A. Tumor cell invasion through matrigel is regulated by activated matrix metalloproteinase-2. Anticancer Res 1997;17:3201–10.[Medline]
  8. Brooks PC, Stromblad S, Sanders LC, et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin {alpha} v ß 3. Cell 1996;85:683–93.[CrossRef][Medline]
  9. Wild-Bode C, Weller M, Rimner A, Dichgans J, Wick W. Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma. Cancer Res 2001;61:2744–50.[Abstract/Free Full Text]
  10. Sonveaux P, Brouet A, Havaux X, et al. Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: implications for tumor radiotherapy. Cancer Res 2003;63:1012–9.[Abstract/Free Full Text]
  11. Qian LW, Mizumoto K, Inadome N, et al. Radiation stimulates HGF receptor/c-Met expression that leads to amplifying cellular response to HGF stimulation via upregulated receptor tyrosine phosphorylation and MAP kinase activity in pancreatic cancer cells. Int J Cancer 2003;104:542–9.[CrossRef][Medline]
  12. Qian LW, Mizumoto K, Urashima T, et al. Radiation-induced increase in invasive potential of human pancreatic cancer cells and its blockade by a matrix metalloproteinase inhibitor, CGS27023 Clin Cancer Res 2002;8:1223–7.[Abstract/Free Full Text]
  13. Onoda JM, Piechocki MP, Honn KV. Radiation-induced increase in expression of the {alpha} IIb ß 3 integrin in melanoma cells: effects on metastatic potential. Radiat Res 1992;130:281–8.[CrossRef][Medline]
  14. Cox R, Thacker J, Goodhead DT, Munson RJ. Mutation and inactivation of mammalian cells by various ionising radiations. Nature 1977;267:425–7.[CrossRef][Medline]
  15. Goodhead DT, Thacker J, Cox R. Non-rejoining DNA breaks and cell inactivation. Nature 1978;272:379–80.[CrossRef][Medline]
  16. Suzuki M, Kase Y, Yamaguchi H, Kanai T, Ando K. Relative biological effectiveness for cell-killing effect on various human cell lines irradiated with heavy-ion medical accelerator in Chiba (HIMAC) carbon-ion beams. Int J Radiat Oncol Biol Phys 2000;48:241–50.[Medline]
  17. Takahashi Y, Teshima T, Kawaguchi N, et al. Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose. Cancer Res 2003;63:4253–7.[Abstract/Free Full Text]
  18. Asai T, Ueda T, Itoh K, et al. Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. Int J Cancer 1998;76:418–22.[CrossRef][Medline]
  19. Kagawa K, Murakami M, Hishikawa Y, et al. Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center. Int J Radiat Oncol Biol Phys 2002;54:928–38.[CrossRef][Medline]
  20. Kanai T, Endo M, Minohara S, et al. Biophysical characteristics of HIMAC clinical irradiation system for heavy-ion radiation therapy. Int J Radiat Oncol Biol Phys 1999;44:201–10.[CrossRef][Medline]
  21. Kanai T, Kohno T, Minohara S, et al. Dosimetry and measured differential W values of air for heavy ions. Radiat Res 1993;135:293–301.[Medline]
  22. Pei D, Weiss SJ. Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J Biol Chem 1996;271:9135–40.[Abstract/Free Full Text]
  23. Suit H, Urie M. Proton beams in radiation therapy. J Natl Cancer Inst 1992;84:155–64.[Abstract/Free Full Text]
  24. Lin R, Slater JD, Yonemoto LT, et al. Nasopharyngeal carcinoma: repeat treatment with conformal proton therapy-dose-volume histogram analysis. Radiology 1999;213:489–94.[Abstract/Free Full Text]
  25. Bush DA, Slater JD, Bonnet R, et al. Proton-beam radiotherapy for early-stage lung cancer. Chest 1999;116:1313–9.[Abstract/Free Full Text]
  26. Yonemoto LT, Slater JD, Rossi CJ Jr, et al. Combined proton and photon conformal radiation therapy for locally advanced carcinoma of the prostate: preliminary results of a phase I/II study. Int J Radiat Oncol Biol Phys 1997;37:21–9.[CrossRef][Medline]
  27. Miyamoto T, Yamamoto N, Nishimura H, et al. Carbon ion radiotherapy for stage I non-small cell lung cancer. Radiother Oncol 2003;66:127–40.[CrossRef][Medline]
  28. Kamada T, Tsujii H, Tsuji H, et al. Efficacy and safety of carbon ion radiotherapy in bone and soft tissue sarcomas. J Clin Oncol 2002;20:4466–71.[Abstract/Free Full Text]
  29. Cordes N, Blaese MA, Meineke V, Van Beuningen D. Ionizing radiation induces up-regulation of functional ß1-integrin in human lung tumour cell lines in vitro. Int J Radiat Biol 2002;78:347–57.[CrossRef][Medline]
  30. Cordes N, Meineke V. Cell adhesion-mediated radioresistance (CAM-RR). Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro. Strahlenther Onkol 2003;179:337–44.[CrossRef][Medline]
  31. Zhang Z, Vuori K, Wang H, Reed JC, Ruoslahti E. Integrin activation by R-ras. Cell 1996;85:61–9.[CrossRef][Medline]
  32. Parise LV, Lee J, Juliano RL. New aspects of integrin signaling in cancer. Semin Cancer Biol 2000;10:407–14.[CrossRef][Medline]
  33. Wang JL, Sun Y, Wu S. {gamma}-Irradiation induces matrix metalloproteinase II expression in a p53-dependent manner. Mol Carcinog 2000;27:252–8.[CrossRef][Medline]
  34. Cordes N, Hansmeier B, Beinke C, Meineke V, van Beuningen D. Irradiation differentially affects substratum-dependent survival, adhesion, and invasion of glioblastoma cell lines. Br J Cancer 2003;89:2122–32.[CrossRef][Medline]
  35. Bian J, Sun I. Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase-2) promoter. Mol Cell Biol 1997;17:6330–8.[Abstract]
  36. Komarova EA, Zelnick CR, Chin D, et al. Intracellular localization of p53 tumor suppressor protein in {gamma}-irradiated cells is cell cycle regulated and determined by the nucleus. Cancer Res 1997;57:5217–20.[Abstract/Free Full Text]
  37. Asakawa I, Yoshimura H, Takahashi A, et al. Radiation-induced growth inhibition in transplanted human tongue carcinomas with different p53 gene status. Anticancer Res 2002;22:2037–43.[Medline]
  38. von Essen CF. Radiation enhancement of metastasis: a review. Clin Exp Metastasis 1991;9:77–104.[CrossRef][Medline]
  39. Baumann M, Becker S, Kruger HJ, Vogler H, Maurer T, Beck-Bornholdt HP. Flow cytometric determination of the time of metastasis during fractionated radiation therapy of the rat rhabdomyosarcoma R1H. Int J Radiat Biol 1990;58:361–9.[Medline]
  40. Ando S, Nojima K, Ishihara H, et al. Induction by carbon-ion irradiation of the expression of vascular endothelial growth factor in lung carcinoma cells. Int J Radiat Biol 2000;76:1121–7.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 JNMHome page
Z.-b. Li, W. Cai, Q. Cao, K. Chen, Z. Wu, L. He, and X. Chen
64Cu-Labeled Tetrameric and Octameric RGD Peptides for Small-Animal PET of Tumor {alpha}v{beta}3 Integrin Expression
J. Nucl. Med., July 1, 2007; 48(7): 1162 - 1171.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogata, T.
Right arrow Articles by Matsuura, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogata, T.
Right arrow Articles by Matsuura, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online