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Experimental Therapeutics |
Department of Urology Osaka Medical College, Takatsuki, Osaka, 569-8686 [H. A., Y. K.]; Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871 [S. T., N. I., J. D. W., N. N., A. O.]; Department of Anatomy and Biology [Y. I., Y. O.], Laboratory Animal Center [J. M.], and First Department of Internal Medicine and Central Research Laboratory [R. F., M. H., T. I.], Osaka Medical College, Takatsuki, Osaka, 569-8686; and Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Setagaya, Tokyo, 154-8509 [S. S.] Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study showed that FTY720 dramatically inhibited in vivo tumor growth with no severe side effects. In addition, it is noteworthy that the treatments with this agent significantly prevented metastasis, which is one of the major causes of mortality in cancer patients. The development of cancer metastasis is a complex cascade of events involving cell-cell interactions between tumor cells and host cells. The formation of tumor aggregates or emboli may be arrested nonspecifically in narrow capillaries; subsequently some of these tumor cells may penetrate the vascular wall, degrade the basement membrane and ECM,2 and then invade the secondary organ (10, 11, 12, 13) . The interaction of tumor cells with ECM components is thought to be critical in such metastatic cascade (14, 15, 16) . In addition, the expression of integrins and adhesion molecules is reported to contribute to the events (14, 15, 16, 17, 18) . Concerning cancer treatment, especially for prevention of metastasis, it may be possible that interference of cell-cell or cell-ECM adhesions by modulating the adhesive elements, such as integrins or ECM components, blocks, or suppresses cancer metastasis. Therefore, we conducted the present study to test the in vivo effect of FTY720 on prevention of cancer growth and metastasis. Furthermore, we investigated its mechanisms of action, especially for preventing metastasis directing on the process of cancer cell arrest.
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MATERIALS AND METHODS |
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Colorimetric (MTT) Assay.
Cell viability after FTY720 treatment was assessed using a Cell Survival and Proliferation kit (Chemicon, Temecula, CA). We plated 100 µl of a cell suspension (5000 cells) in each well of a 96-well flat-bottomed microtiter plate and incubated them overnight at 37°C in a humidified 5% CO2 atmosphere. We then added FTY720 in each well at the various concentrations (10, 30, 50, and 100 µM) by substituting new culture medium containing the drug at each concentration. After the incubation with FTY720 for 2 h, we added 10 µl of MTT prepared at a concentration of 5 mg/ml in PBS to each well and continued the cell culture for another 4 h at 37°C. We then added a color development solution to each well and measured absorbance using a microculture plate reader with a wavelength of 570 nm. Cell viability was expressed as a percentage of absorbance in cells with FTY720 treatment to that in cells without FTY720 treatment.
Flow Cytometry.
JygMC(A) cancer cells and NCTC-2525 fibroblasts were synchronized at the G0/G1 phase by the double thymidine block method as follows. The cell number was adjusted to 5 x 105 for plating in fresh medium before the procedure. Cells were incubated with fresh medium containing 2.5 mM of thymidine without FCS for 12 h and then washed twice with PBS, followed by addition of fresh medium with 10% FCS and additionally incubated for another 12 h. After this, we again incubated cells with fresh medium containing 2.5 mM of thymidine without FCS for 12 h. To release the cells from the block, we washed the cells twice with PBS and replated them in fresh medium with 10% FCS. After synchronization, we incubated the cells with or without 10 µM of FTY720 for 3, 6, 9, 12, 18, or 24 h. The cells were harvested after treatment with 0.2% EDTA, fixed with ice-cold 70% ethanol solution, hydrolyzed with 250 µg/ml of RNaseA (type 1-A; Sigma Chemical Co.) at 37°C for 30 min, and stained with propidium iodide (Sigma Chemical Co.) at 10 mg/ml for 20 min. We analyzed the DNA content of the cells by an EPICS ELITE flow cytometer (Coulter, Hialeah, FL) and calculated the cell cycle distribution using a MULTICYCLE program (Phoenix Flow Systems, San Diego, CA).
Cell Growth Assay.
JygMC(A) cancer cells and NCTC-2525 fibroblasts were synchronized at the G0/G1 phase by the double thymidine block method as described above. After synchronization, we incubated the cells with or without FTY720 at the various concentrations (2, 5, 10 15, 30, and 50 µM) for 24 h. The number of cells was determined after the incubation with FTY720, and cell growth rate was expressed as the percentage of the cell number in the wells treated with FTY720 compared with that in the control wells.
Electron Microscopic Analysis.
For electron microscopic observation, the cancer cells were incubated with FTY720 at the various concentrations (2, 5, and 10 µM) for 24 h. The cells cultured without FTY720 served as controls. We harvested floating cells together with adherent cells and centrifuged them at 2000 rpm for 5 min. The cell pellets were fixed overnight at 4°C in a 0.2 M sodium cacodylate buffer containing a 2% solution of glutaraldehyde. Samples were then postfixed in cacodylate-buffered 1% osmium tetroxide, dehydrated, and embedded in Epon 812 (Nacalai Tesque, Osaka, Japan) for ultrathin sectioning. We stained the ultrathin sections with uranyl acetate and lead citrate, and viewed them with an electron microscope (H7100; Hitachi, Tokyo, Japan).
Fluorecent Staining of F-Actin with FITC-labeled Phalloidin.
Fluorecent staining of F-actin was carried out according to the method of Sasaki et al. (20)
with minor modification. JygMC(A) cells were plated on 8-mm square Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) and incubated with FTY720 at the various concentrations (2, 5, 10, and 30 µM) for 2 h at 37°C in a CO2 incubator. The cells were washed with PBS, fixed in a 4% paraformaldehyde-PBS solution, permeabilized with 0.5% Triton X-100, and stained with FITC-labeled phalloidin. The slides were analyzed by a fluorescence microscope (UFX-DX; Nikon, Tokyo, Japan).
Adhesion Assay.
We examined the influence of FTY720 on ability of JygMC(A) cancer cells to adhere to ECM components including laminin, collagen I, and fibronectin. Briefly, we coated a 96-well culture plate with laminin (50 µg/ml; Sigma Chemical Co.), collagen I (300 µg/ml; Chemicon), or fibronectin (20 µg/ml; Sigma Chemical Co.), and blocked the plate with 10 mg/ml BSA. JygMC(A) cells (2 x 105) with or without 3-hour incubation of FTY720 at a concentration of 2 µM were added to each well. After another 3-hour incubation at 37°C, the cells were washed twice with PBS to remove nonadherent cells and fixed with 4% of paraformaldehyde solution for 10 min. Samples were stained with toluidine blue and rinsed in water. Cells were then solubilized by adding 1% SDS and quantified using a microtiter plate reader at 595 nm. The data were represented as absorbance; cells treated with FTY720 and control cells were then compared. The experiments described were repeated a minimum of three times.
Migration Assay.
We performed cell migration assay by a modified Boyden’s chamber method using microchemotaxis chambers and polycarbonate filters (Neuro Probe, Gaithersburg, MD) with a pore size of 8.0 µm. The filters were coated with 20 µg/ml fibronectin, 50 µg/ml laminin, or 300 µg/ml collagen I, and placed between upper and lower chambers. We next prepared cell suspensions of JygMC(A) cells (5 x 105) with or without treatment of FTY720 at 2 µM for 3 h in serum-free DMEM containing BSA. We then placed the cells (2.5 x 104 cells in 50 µl) into the upper compartment of chamber and placed 25 µl of DMEM containing 10% FCS in the lower chamber. Chambers were incubated at 37°C with 5% CO2 for 3 h. Thereafter, we removed the filter and scraped off nonmigrating cancer cells remaining on the upper side of the filter. The cells that had migrated to the lower side of the filter were fixed in methanol, stained with toluidine blue, and counted in >10 fields of view under a microscope at x 200 magnification. Migratory activity was expressed as the mean number of cells that had migrated to the lower side of the filter, and results were represented as mean ± SD of cells per field of view. The experiments described were repeated a minimum of three times.
Flow Cytometric Analysis for Integrin Expression.
JygMC(A) cells (2 x 105) cultured in DMEM containing 10% FCS were treated with or without FTY720 at a concentration of 2 µM for 6 h. The cells were harvested after treatment with 0.2% EDTA. After fixation with 4% of paraformaldehyde solution, the cells were incubated with 10-fold dilution of goat antimouse polyclonal antibodies against the following integrins: VLA1 (
1ß1), VLA2 (2ß1), VLA5 (5ß1), and VLA6 (6ß1; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The cells were then incubated with an 80-fold dilution of FITC-conjugated donkey antigoat IgG (Santa Cruz Biotechnology) at 4°C for 30 min. After each incubation, the cells were washed three times with PBS containing 1% BSA and analyzed with an EPICS ELITE flow cytometer (Coulter).
In Vivo Effect of FTY720 on Tumor Growth in a Mouse Breast Cancer Model.
We studied the in vivo effect of FTY720 in preventing tumor growth using a mouse breast cancer model (Group 1). Aliquots of JygMC(A) cancer cells (1 x 107) were s.c. inoculated into BALB/c-nu/nu mice. The mice were housed in microisolator cages under positive air pressure, and maintained at a constant temperature (20°C) and humidity. Surgical manipulations and drug treatments were performed in a laminar flow cabinet. When the tumors reached 
10 x 10 mm in size, the animals were assigned randomly to four subgroups, and the treatment with FTY720 was initiated at the three different dosages. Mice from three subgroups received an i.p. injection of FTY720 daily at a dosage of 2 mg/kg (Gp1–2; n = 30), 5 mg/kg (Gp1–5; n = 30), or 10 mg/kg (Gp1–10; n = 30); drug administration was continued until the end of the study. Mice without FTY720 treatment served as controls (Gp1-Cont; n = 90). Tumor size was measured every 3 days in two dimensions, the longest axis (a) and perpendicular shortest axis (b). TV was calculated using the formula TV = 0.4ab2 and transformed into relative values (v) (v = Vx/V0, where V0 is the TV at initiation of treatment and Vx is the volume at any given day). Animals were sacrificed on days 2, 24, and 35, at which times tumors and tissues were harvested and prepared for morphological and molecular analysis.
The Influence of FTY720 on Tumor Metastasis In Vivo.
Because the present animal model aggressively develops numerous metastatic lesions in the lung, liver, and kidney, we investigated the influence of FTY720 on tumor metastasis using this model. We set up another experimental group (Group 2) in which tumors were excised from mice when the tumors reached 10 x 10 mm in size to eliminate the influence of TV on the development of cancer metastasis. Animals were randomly assigned to four subgroups: mice not treated with FTY720 (Gp2-Cont), and mice treated with FTY720 i.p. at a daily dosage of 2 mg/kg (Gp2–2; n = 30), 5 mg/kg (Gp2–5; n = 30), or 10 mg/kg (Gp2–10; n = 30). The drug administration was initiated immediately after tumor excision and continued until the end of the study. Animals were sacrificed on day 24 after initiation of drug administration, and harvested organs were weighed and fixed with 4% buffered formalin after perfusion. The paraffin sections were stained with H&E for light microscopic assessment. The number of metastatic colonies in each organ was counted in a histological section. For more accurate quantitative evaluation of cancer metastasis, the ratio of metastatic area:total area in a histological section was calculated using a public domain NIH image program (written by Wayne Rasband at NIH and available on the internet3
) and MCID program (Imaging Research Inc., Ontario, Canada). The results were represented as a percentage of metastatic area:total area ratio.
In Situ Assay for DNA Fragmentation.
We observed in situ DNA fragmentation in the tumor sections with the TUNEL method. The sections were prepared as described previously (21)
. Staining for DNA fragmentation was performed using an In Situ Apoptosis Detection kit (Takara; Nihonbashi, Tokyo, Japan), according to the manufacturer’s instructions.
Transmission Electron Microscopy of Tumors from Mice.
For electron microscopic observation, tumors were fixed, and ultrathin sections were prepared as described above and viewed with an electron microscope (H7100; Hitachi).
Ethics.
All of the experimental protocols were conducted in accordance with the policies of the Animal Ethics Committee at our institution.
Statistical Analysis.
The number of filopodias on the cell surface, the data from adhesion assay and migration assay, and the expression of integrins were subjected to the unpaired Student’s t test. Results from TV were subjected to one-way ANOVA without replication. When the ANOVA resulted in significance, individual comparisons were performed by the Student t test. The unpaired Student’s t test was applied to values obtained for quantitative evaluation of tumor metastasis including organ weight, number of metastatic colonies, and the ratio of metastatic area:total area. Animal survival was evaluated by Kaplan-Meier analysis. P < 0.05 was considered to be statistically significant.
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RESULTS |
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. This was confirmed by flow cytometric analysis where >80% of cancer cells died within 12 h after the initiation of FTY720 treatment at 10 µM, whereas the cells without FTY720 treatment demonstrated normal cell viability with no remarkable cell death (Fig. 1B)
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. Furthermore, the drug susceptibility to FTY720 was significantly lower in the normal mouse fibroblasts than in the cancer cells. Treatment with FTY720 at a concentration of 2 µM did not affect the cell viability in NCTC-2525, which exhibited normal cell proliferation (Fig. 2A)
. Although a decreased cell growth rate was noted in NCTC-2525 with the treatment of FTY720 at a concentration of 5 µM, the effect of FTY720 was significantly less in NCTC-2525 than in the cancer cells. These results were confirmed by flow cytometric analysis; only 16.7 ± 5.8% of NCTC-2525 cells underwent apoptosis at 24 h after FTY720 treatment at 5 µM, whereas 77.8 ± 4.68% of cancer cells underwent apoptosis in the same condition (Fig. 2B)
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. However, cancer cells treated with FTY720 at a low concentration of 2 µM demonstrated distinct changes on the cell surface with decreased filopodias and microvilli, despite no remarkable changes being noted in the nuclei (Fig. 3b)
. In contrast, FTY720 treatment at 10 µM induced features typical of apoptosis including fragmented nuclei with condensed chromatin accompanied by well-preserved cytoplasmic organellas, such as mitochondrias (Fig. 3c)
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; Fig. 4
). When cancer cells were treated with the drug at 2 µM, cells became relatively round with shortened stress fibers and deformed filopodias on the cell surface as compared with cells without FTY720 treatment. The stress fibers were markedly shortened, and cells became round with remarkably decreased and flattened filopodias at 10 µM. The filamentous actin structure was totally disrupted with marked deformity in cancer cells at 30 µM.
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. In addition, we investigated the migration activity of cancer cells using a modified Boyden’s chamber method. The cancer cell migration was significantly suppressed after 6 h of FTY720 treatment at 2 µM, particularly when filters were coated with laminin (Fig. 5B)
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1ß1), VLA2 (2ß1), VLA5 (5ß1), and VLA6 (6ß1) that are known to be critical for cancer metastasis, was determined by flow cytometric analysis. As shown in Fig. 6
, 6-h FTY720 treatment at 2 µM significantly decreased the expression of integrins, especially VLA1, VLA2, and VLA6, which bind to laminin and collagen type I.
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shows the time course of tumor growth in mice. Treatment with FTY720 at 2 mg/kg/day did not show any significant effect in preventing tumor growth.
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. In contrast, neoplastic glands showed structural deformity and collapse with characteristic signs of apoptosis 2 days after initiation of FTY720 treatment at a dosage of 
5 mg/kg/day. Affected cells appeared round, and contained highly condensed nuclear chromatin and fragmented nuclei (Fig. 8b)
. Visualization of DNA fragments using the TUNEL method clearly corroborated these findings, showing many positive cells in the tumors from FTY720-treated mice (Fig. 8d)
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. In contrast, the tumor cells treated with FTY720 exhibited features characteristic of apoptosis, including fragmented nuclei with condensed chromatin and cell shrinkage (Fig. 8f)
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Prevention of Cancer Metastasis in Lung, Liver, and Kidney by Treatment with FTY720.
In control groups (n = 90/group), >90% of the mice developed numerous metastatic lesions in the lung, and >50% of the mice developed metastasis in the liver and kidney by 3 weeks; these progressed aggressively and diffusely thereafter. Fig. 9a
presented the in situ appearance of a mouse sacrificed at 5 weeks after inoculation of cancer cells. The mouse exhibited numbers of metastatic lesions in the lung, liver, and kidneys. In contrast, when mice were treated with FTY720, no evident metastasis was observed in the lung, liver, and kidneys (Fig. 9b)
; only a few cancer cells were noted in the histological section of each organ even at a dosage of 2 mg/kg/day at which dose the treatment did not show any preventive effect on tumor growth (Fig. 9c)
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10 x 10 mm in size. In the control group, most of the mice developed numbers of metastatic lesions in the lung, liver, and kidney by 3 weeks after the resection of tumors. Fig. 10 (a–c)
shows numerous metastatic lesions as a hematoxylin-stained area in H&E sections of each organ. These lesions occupy more than half of the total area especially in the lung sections. Microscopic findings showed aggressive cancer growth with innumerable mitosis, which replaced normal organ construction and caused structural deformity in each organ (Fig. 10, a–c)
. In contrast, when mice were treated with FTY720, no remarkable metastatic lesions were observed macroscopically (Fig. 10, g–j)
, and only a few cancer cells were noted in the histological sections 24 days after resection of tumors (Fig. 10, d–f)
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. The weight of organs harvested from FTY720-treated mice even at a low dose (2mg/kg/day) significantly decreased and correlated with a reduced number of metastatic colonies as compared with those from control mice. The ratio of metastatic area:total area confirmed the data, demonstrating a significant decrease in the metastatic area in the organs from FTY720-treated mice (Table 2)
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. In contrast, when mice were treated with FTY720, survival was significantly prolonged in all of the subgroups (Gp1–2, Gp1–5, and Gp1–10) as compared with control group. No animal died before 38 days, and >50% of the animals survived >52 days.
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DISCUSSION |
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Indeed, the present study showed that FTY720 powerfully inhibited in vivo tumor growth in a mouse model of breast cancer while causing no severe side effects. In these animals, histological sections of tumors stained by H&E and TUNEL methods demonstrated the characteristic features of apoptosis in many cancer cells. Apoptosis was confirmed by electron microscopy, affected cells exhibiting small cytoplasm and fragmented nuclei with condensed chromatin, and some exhibiting crescent formation. Despite such dramatic cancer cell apoptosis, no remarkable side effects were found in mice treated with FTY720, suggesting that cancer cells are more susceptible to FTY720 than normal cells.
In vitro treatment of cancer cells corroborated these results. MTT and cell growth assays demonstrated a dramatic loss of viability of cancer cells with treatment of FTY720 in a dose-dependent fashion. The flow cytometric analysis showed that the initiation of drastic cancer cell death occurred by 9 h after incubation of FTY720, and most of cancer cell death was observed by 24 h. Morphological study by light and electron microscopy revealed typical changes of apoptosis after treatment with FTY720. Interestingly, the effect of FTY720 was significantly less in the normal fibroblasts than in cancer cells. In a previous study, we also found that FTY720 rapidly induced apoptosis in a human prostate cancer cell line, DU145, whereas normal human prostate stromal cells were resistant to FTY720 (22) . These findings indicate that cancer cells are more susceptible to FTY720.
The mode of action of most currently used chemotherapeutic agents is based on the preferential toxicity to rapidly dividing cells, e.g., malignancies that have relatively rapid growth, such as germ cell tumors, are relatively chemosensitive, whereas slower growing neoplasmas such as renal cell carcinoma are less sensitive. Similarly, toxicity from chemotherapeutic agents in normal, nonmalignant cells is also seen primarily in rapidly dividing cells, such as hematopoietic cells in the bone marrow, especially granulocyte, which has very high proliferation activity. Therefore, a reduction of granulocytes, which causes the most serious side effect, is frequently seen in patients treated with current chemotherapeutic agents. In contrast to these chemotherapeutic agents, FTY720 may exhibit its activity through a different mechanism. In our previous and present studies, the agent induced a drastic apoptosis in peripheral lymphocytes, whereas no remarkable change was noted in other hematopoietic cells, including granulocytes, megakaryocytes, and erythrocytes (5 , 6) . These findings may indicate that the drug-induced apoptosis is not based on the cell toxicity, but it is possible that FTY720 activates a certain intracellular pathway of apoptosis, which may exist commonly both in cancer cells and lymphocytes. We have investigated the intracellular mechanisms of action of this agent in detail, providing the following evidence up to now. The intracellular ratio of Bcl-2:Bax proteins in the FTY720-exposed cultured human lymphocytes decreased immediately after treatment with FTY720 (4) . This suggests that the drug induces Bcl-2-family associated apoptotic cell death. In addition, Fas-mutant mice (MRL-lpr/lpr) treated in vivo with FTY720 exhibited a widespread lymphocyte apoptosis in the thymus, spleen, and lymph nodes (23) , indicating that the drug-induced cell death occurs via a Fas-independent pathway. Furthermore, recent studies have shown that the drug induces activation of caspase cascade (7) and that apoptosis is related to protein kinases, including c-Jun-NH2-terminal kinase, p38, and a novel Mr 36,000 myelin basic protein (8) . Although additional investigation is necessary, FTY720 may be a new cancer-targeting agent, which induces cancer cell death by promoting the apoptotic signal pathways.
Additionally, one of most important findings in this study is the marked prevention of tumor metastasis by FTY720 treatment even at a low dose. In control groups, most of the mice developed numerous metastatic lesions in the lung, liver, and kidney. Histological sections of these organs showed aggressive cancer growth accompanied by innumerable mitosis, which replaced normal organ tissue. In contrast, mice treated with FTY720, even at a low dose of 2 mg/kg/day, exhibited normal macroscopic appearance in the lung, liver, and kidney, although the agent did not influence tumor growth at this dosage. Only a few cancer cells were noted in the histological sections of the distant organs from these animals.
Although metastasis is one of the major causes of mortality in cancer patients, there are few therapeutic options for patients with advanced disease and metastases. The development of cancer metastasis is a complex cascade of events involving tumor dissemination from the primary site to distant organs. The cancer cells released from the primary tumor invade the surrounding tissues, enter into the vascular or lymphatic circulation (10 , 11 , 24) , and then become arrested on the distant organ. The formation of tumor-tumor aggregates or emboli is an important event in this step (13) . Tumor emboli may be arrested nonspecifically in narrow capillaries; subsequently, some of the tumor cells may penetrate the endothelium, degrade the basement membrane and ECM, and invade the secondary organ.
In the present study, the data from the staining of F-actin indicated that FTY720 caused remarkable cytoskeletal change with a marked disorganization of actin stress fiber, such as a decrease in number and length of actin-containing microfilaments in a dose-dependent manner. In addition, cells became round with decreased and deformed filopodias on the cell surface even at a low dose of FTY720 treatment. Electron microscopic findings confirmed these changes, revealing round cells with decreased filopodias and loss of microvilli on the cell surface. Such structural changes in cancer cells may contribute to the prevention of the metastatic cancer dissemination through the interruption of cancer cell arrest within the target organs.
After arrest, tumor cells must establish stable contacts with the endothelium, induce endothelial cell retraction, migrate, and attach to several connective tissue barriers that consist of ECMs such as laminin, fibronectin, and other glycoproteins and proteoglycans. Finally, they must proteolytically degrade these connective tissues and extravasate. During this process, the interaction of tumor cells with ECM components is thought to be mostly dependent on the presence of integrins (14 , 17) . The altered integrin-mediated adhesion of cancer cells to ECMs has been reported to be a critical factor in the metastatic cascade (15 , 16 , 18 , 25) ; e.g., transfection of VLA-2 cDNA into rhabdomyosarcoma cells resulted in marked increase of adhesion to collagen and laminin in vitro and enhancement of lung metastasis in vivo (26) . Cancer cell transformation is reported to be implicated in the altered expression of integrins (27, 28, 29, 30, 31) . Human cells subjected to N-methyl-N'-nitro-N-nitrosoguanidin transformed into highly tumorigenic cells with a significant increase in the expression of the integrins such as VLA1, VLA2, and VLA6 that promote the invasive ability through the basement membranes (29) . The present study demonstrated that FTY720 treatment at a low concentration significantly decreased the ability of cancer cells to adhere to ECM components, especially laminin. In addition, the activity of cancer cell migration through ECMs was also significantly reduced by the treatment at a low concentration, especially when filters were coated with laminin. The results indicate that FTY720 treatment at a low concentration may decrease the ability of cancer cells to interact with ECM components. Furthermore, the expression of integrins, especially VLA1, VLA2, and VLA6, that are the ligands for laminin and collagen type I, was significantly decreased in cancer cells treated with FTY720. These findings suggest that the reduced expression of integrins may be implicated with a decreased ability of cancer cells to interact with ECM components, which is thought to be a critical process for the development of cancer metastasis.
In conclusion, the present study has demonstrated that FTY720 displays a potent anticancer activity by inducing apoptosis in vitro and in vivo. Additionally, it prevents dissemination of cancer metastases in animal models even at a lower dosage. Although additional investigation is necessary, the decreased ability of cancer cells to interact with ECM components, which is caused by FTY720 treatment, may be associated with the mechanism of the prevention of the cancer metastasis. Structural changes in cancer cells and decreased expression of integrins on the cell surface is possibly one of its underlying mechanisms of action. Thus, FTY720 may be a promising agent for the treatment of cancer patients even if they demonstrate evidence of metastatic spread of cancer.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3531; Fax: 81-6-6879-3539; E-mail: takahara{at}uro.med.osaka-u.ac.jp. 
2 The abbreviations used are: ECM, extracellular matrix; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Gp, group; TV, tumor volume; TUNEL, terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling; VLA, very late antigen; F-actin; filamentous actin.
3 Internet address: http://info.nih.gov/nih-image/download.htm.
Received 8/22/01. Accepted 1/ 4/02.
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 T. K. Lee, K. Man, J. W. Ho, X. H. Wang, R. T. Poon, C. K. Sun, K. T. Ng, I. O. Ng, R. Xu, and S. T. Fan Significance of the Rac signaling pathway in HCC cell motility: implications for a new therapeutic target Carcinogenesis, March 1, 2005; 26(3): 681 - 687. [Abstract] [Full Text] [PDF]
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T. K. Lee, K. Man, J. W. Ho, C. K. Sun, K. T. Ng, X. H. Wang, Y. C. Wong, I. O. Ng, R. Xu, and S. T. Fan FTY720 induces apoptosis of human hepatoma cell lines through PI3-K-mediated Akt dephosphorylation Carcinogenesis, December 1, 2004; 25(12): 2397 - 2405. [Abstract] [Full Text] [PDF]
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S. Majstoravich, J. Zhang, S. Nicholson-Dykstra, S. Linder, W. Friedrich, K. A. Siminovitch, and H. N. Higgs Lymphocyte microvilli are dynamic, actin-dependent structures that do not require Wiskott-Aldrich syndrome protein (WASp) for their morphology Blood, September 1, 2004; 104(5): 1396 - 1403. [Abstract] [Full Text] [PDF]
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