Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis Kazumoto Murata1 and Masami Moriyama2

Research Article

1The First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan and 2Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan

Abstract infection in the airways of cystic fibrosis (8), and susceptibility to salmonella infection in mouse intestinal tracts (9). In addition to In spite of recent advances in the treatment of colon cancer, h multiple liver metastases of colon cancer are still difficult to their direct antimicrobial activities, -defensins are strong treat. Some chemotherapeutic regimens have been reported to chemotactic factors for memory T cells and dendritic cells, be efficient, but there is a high risk of side effects associated suggesting that they also play an important role in acquired immunity (10–12). h-defensins are also inducible by inflammatory with these. Here, we show that isoleucine, an essential amino a h acid, prevents liver metastases in a mouse colon cancer cytokines, such as tumor necrosis factor- and interleukin-1 (13, 14). Recently, Fehlbaum et al. (15) reported that isoleucine metastatic model. Because isoleucine is a strong inducer of h B-defensin, we first hypothesized that it prevented liver and its analogues are highly specific inducers of -defensins. We thus originally hypothesized that isoleucine may contribute to metastases via the accumulation of dendritic cells or memory B tumor immunity through both innate and acquired immunity by T cells through up-regulation of -defensin. However, neither h B-defensin nor immunologic responses were induced by induction of -defensins. isoleucine in both mouse livers and spleens. Furthermore, Angiogenesis is a key process in tumor growth (16–18), and isoleucine prevented liver metastasis in nude mice, which lack vascular endothelial growth factor (VEGF), which stimulates T cells and natural killer T cells. Finally, we discovered a novel angiogenesis in a paracrine fashion, seems to be essential for the mechanism of isoleucine: down-regulation of angiogenesis via progression of various solid tumors. VEGF production is initiated inhibition of vascular endothelial growth factor, partially by phosphorylation of eukaryotic initiation factor (eIF) 4E-binding through the mammalian target of the rapamycin pathway, protein 1 (4E-BP1) by the mammalian target of the rapamycin independent of hypoxia-inducible factor 1-A. Importantly, (mTOR) following disruption of their binding to eIf-4E, enabling eIF-4E to translate most mRNAs, including VEGF, through the isoleucine is safe for administration to humans because it does not affect cell viability. Isoleucine could be a novel m7GpppN cap (19–21). Rapamycin, an immunosuppressive drug, prophylactic drug for the prevention of liver metastases of interacts with mTOR, following dephosphorylation of 4E-BP1 and colon cancer. [Cancer Res 2007;67(7):3263–8] impairment of VEGF production (22). Recent studies have shown that leucine promotes albumin production partially through the mTOR pathway, which stimulates VEGF through activation of Introduction eIF-4E (23, 24). However, the association between isoleucine and Colorectal cancer is one of the leading causes of deaths from angiogenesis remains unclear. cancer worldwide. In Japan, the incidence of colorectal cancer Here, we describe remarkable results indicating that isoleucine has increased in association with diets becoming more ‘‘West- actually prevents tumor growth in a mouse colon cancer liver ernized.’’ In spite of recent advances in the treatment of colon metastatic model. The mechanism by which isoleucine exerts its cancer, the prognosis of patients with multiple liver metastases of antitumor effect was not, as we expected, via an immunologic colon cancers is still poor, and systemic chemotherapy prolongs pathway through the up-regulation of h-defensins. Rather, survival only by a few months (1). Moreover, there is a high risk isoleucine acts through a novel mechanism whereby it inhibits of severe side effects (2, 3), although methods for reducing the VEGF at the translation initiation level, partially through the side effects of systemic chemotherapy [e.g., infusion chemother- mTOR pathway. Importantly, isoleucine did not affect cell apy through a reservoir pump (4) or chronotherapy (5)] can be viability. used. Antimicrobial peptides, such as defensins, play an important role in the innate immunity of all life forms, including plants or Materials and Methods insects, which lack immune systems comprising such elements as Animals and cell lines. Male BALB/c mice (6–8 weeks old) and male antibodies or lymphocytes. Several types of antimicrobial peptides BALB/c nude mice (nu/nu, 6–8 weeks old) were obtained from SLC are induced at epithelial surfaces in response to inflammation. (Shizuoka, Japan) and maintained with ad libitum access to food and water. These antimicrobial peptides exhibit a broad spectrum of Colon 26 cells (BALB/c syngenic colon cancer cells) were maintained antifungal, antibacterial (6), and antiviral activities (7). Impair- in DMEM (Sigma, Tokyo, Japan) containing 10% FCS. Yac-1 cells ment of the function of defensin leads to susceptibility to were maintained in 10% FCS containing RPMI 1640 (Invitrogen Corp., Carlsbad, CA). Liver metastatic model. Isoleucine, leucine (3 mg/d dissolved in 0.2 mL PBS), or PBS alone was given daily to BALB/c mice or nude mice using a Requests for reprints: Kazumoto Murata, The First Department of Internal feeding tube, starting from 5 days before the tumor implantation until Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie, Japan 514-8507. sacrifice. On the operation day, 1 Â 104 colon 26 cells in 0.2 mL PBS were Phone: 81-59-231-5015; Fax: 81-59-231-5201; E-mail: [email protected]. I2007 American Association for Cancer Research. implanted into the spleens of anesthetized and laparotomized mice. Three doi:10.1158/0008-5472.CAN-06-3739 weeks after the operation, the mice were sacrificed and examined for liver www.aacrjournals.org 3263 Cancer Res 2007; 67: (7). April 1, 2007

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Cancer Research metastases. Blood samples were taken by retro-orbital puncture and serum Results albumin concentrations were measured using a standard clinical automatic analyzer. The liver of each mouse was weighed. Livers, spleens, and tracheas Effect of isoleucine on liver metastasis of colon cancer. We h were removed and frozen until they were used for reverse transcription-PCR first hypothesized that because isoleucine induces -defensins, it (RT-PCR) and Western blotting studies. could contribute to tumor regression through innate or acquired Immunologic experiments. Seven days after administration of immunity. Thus, we attempted to determine whether isoleucine isoleucine or leucine (3 mg/d; n = 5 each), mice were sacrificed and prevents tumor growth in a mouse liver metastatic model of colon intrahepatic lymphocytes were prepared as reported previously (25). The cancer. Remarkably, none of the isoleucine-treated mice (3 mg/d) intrahepatic lymphocytes and splenic lymphocytes were stained with anti- had any liver metastases (n = 5). In contrast, no tumor growth CD3 and anti-panNK(anti-DX5; BD Biosciences, San Jose, CA). The inhibition was observed in the leucine-treated mice (n = 5), relative intrahepatic lymphocytes and splenic lymphocytes were subjected to a to mice treated with PBS (data not shown). These effects were standard natural killer (NK) cytotoxic assay. Briefly, various numbers of intrahepatic lymphocytes and splenic lymphocytes were cultured with 3,000 observed with a rather low concentration of isoleucine 51Cr-coated Yac-1 cells for 5 h. The amount of 51Cr in the supernatant (0.6 mg/d), and the effect on the number of liver metastases was was measured. The CTL concentration was measured using a standard dose dependent (Fig. 1A and B). The effects of isoleucine against 5-h chromium releasing assay. Briefly, splenocytes (nontumor site) from the liver metastases were supported by observations with respect to isoleucine-treated mice (3 mg/mL; n = 5) 3 weeks after implantation of both liver weight (Fig. 1C) and serum albumin levels (Fig. 1D). colon 26 cells or naive mice were incubated with irradiated (10,000 rad) Interestingly, tumor cells in the spleen were found to be growing colon 26 cells in complete culture medium with 10% T-stim (Becton even in the isoleucine-treated mice (3 mg/d), which did not have Dickinson, Billerica, MA) for 7 days following coculture with chromium- any liver metastases. Furthermore, the sizes of the tumors in the coated colon 26 cells for 5 h. The amount of chromium released into the spleen in the isoleucine-treated mice were similar to the sizes of medium was measured. Specific killing was calculated using the following equation: (experimental release À spontaneous release) / (maximum release those in the leucine-treated mice (data not shown). À spontaneous release) Â 100. Isoleucine does not induce mBD-3 in mice. Because h In vitro experiments. Two days after seeding of colon 26 cells at a cell isoleucine is a strong inducer of -defensins (15), we initially density of 1 Â 104 cells/cm2 to a 24-well plate, the medium was replaced hypothesized that h-defensins would be induced in the liver by with isoleucine or leucine dissolved in 2.5% FCS containing DMEM, and the isoleucine treatment. Of the many h-defensins, we focused on cells were cultured for another 48 h. The supernatant was used to measure mBD-3 because human h-defensin-2, which is the homologue of the VEGF concentration by ELISA (mouse VEGF ELISA kit; R&D Systems, mouse BD-3, causes the accumulation of dendritic cells or memory Minneapolis, MN). These cells were used for 3-(4,5-dimethylthiazol-2-yl)-2,5- T cells around tumors and contributes to tumor immunity (10). diphenyltetrazolium bromide (MTT) assay as well for checking for any Additionally, mBD-2 and mBD-3 are chemotactics for immature effects of the amino acids on cell viability. For RT-PCR, cells were harvested dendritic cells (12) and mBD-3 is inducible in the liver (26). 24 h after replacement of the medium. RT-PCR. RT-PCR was used to measure the concentration of mouse Therefore, using RT-PCR, we examined mBD-3 mRNA expression in h-defensin (mBD)-3 and VEGF in the mouse tissues or in the colon 26 cells. the livers and the spleens of mice treated with isoleucine or leucine. Poly(A)+RNA was isolated using RNA Zol (Biotecx Laboratories, Houston, However, no expression of mBD-3 was observed in either liver or TX) from mouse tissues (liver, spleen, and trachea) or cells and a 1 Ag spleen tissue, even in mice, in which liver metastases were aliquot was reverse transcribed into cDNA (Taqman; Applied Biosystems, prevented by isoleucine treatment, whereas mBD-3 mRNA was Foster City, CA). The following primers were used for PCR: BD-3, clearly detected in trachea tissue (positive control; Fig. 2). Thus, it ¶ ¶ ¶ 5 -CTCTTTGCATTTCTCCTGGTGCTGCTG-3 (forward) and 5 -CATCTT- seems that the expression of mBD-3 does not contribute to the ¶ ¶ CATGGAGGAGCAAATTCTG-3 (reverse); VEGF, 5 -GCGGGCTGCC- prevention of liver metastases by isoleucine treatment in our TCGCAGTC-3¶ (forward) and 5¶-TCACCGCCTTGGCTTGTAC-3¶ (reverse). model. RT-PCR using primers specific for mouse glyceraldehyde-3-phosphate dehydrogenase was used as a positive control. Trachea tissue was used as Isoleucine does not induce innate or acquired immunity in a positive control for mBD-3 mRNA. liver or spleen. To investigate the immunologic processes Western blotting. The cell extracts were resolved by SDS-PAGE and participating in the prevention of liver metastases of colon cancer transferred to nitrocellulose membranes. After blocking, the membranes by isoleucine, NKcytotoxicity was examined. NKcytotoxicity was were incubated overnight at 4jC with antibodies against hypoxia- not induced in either the intrahepatic lymphocytes (3.8 F 2.1%; E:T, inducible factor 1-a (HIF1-a; Santa Cruz Biotechnology, Santa Cruz, CA) 50:1) or the splenocytes (6.9 F 5.1%; E:T, 50:1) of the isoleucine- 37/46 or phosphospecific antibodies against 4E-BP1 (Thr ; Cell Signaling treated mice, implying that neither NKnor NKTcells have an Technology, San Diego, CA) and signal transducers and activators of important role in the prevention of liver metastases (naive mice; transcription 3 (Stat3; Santa Cruz Biotechnology). Antibodies against a- intrahepatic lymphocytes, 5.2 F 0.3%; splenocytes, 8.3 F 1.8%, tubulin were used as an internal control. The membranes were washed and then incubated for 30 min with horseradish peroxidase–conjugated respectively). Furthermore, no significant differences in the secondary antibodies. The membranes were washed again, and the populations of intrahepatic lymphocytes or splenocytes were proteins were detected using electrochemiluminescence techniques observed between the isoleucine-treated mice and naive mice (Pierce Chemicals, Rockford, IL). (data not shown). A CTL assay using splenocytes from the Immunohistochemical staining for CD31. Immunohistochemistry isoleucine-treated mice without any liver metastases showed that was done using a peroxidase-streptavidin method on frozen livers, using no specific killing of colon 26 cells was occurring (isoleucine, antibodies against mouse CD31 (BD PharMingen, San Diego, CA). The 5.9 F 4.9%; leucine, 7.0 F 2.1%, respectively; E:T, 50:1). Furthermore, ¶ antibodies were detected with 3,3 -diaminobenzidine. The sections were isoleucine (3 mg/d) completely prevented liver metastases even in counterstained with 10% hematoxylin (Wako Pure Chemical, Osaka, nude mice (n = 5), implying that neither T cells nor NKT cells have Japan). Statistical analysis. Values are expressed as means F SD. Multiple an important role in the prevention of liver metastases by comparisons were done using one-way ANOVA. Intergroup comparisons isoleucine (Fig. 1). Taken together, these results show that neither were done using Bonferroni’s correction for multiple comparisons. A level of innate nor acquired immunity plays a major role in the prevention P < 0.05 was considered statistically significant. of metastatic liver tumors by isoleucine.

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Figure 1. Preventive effect of isoleucine but not leucine on liver metastases of colon cancer cells in mice. After pretreatment with isoleucine or leucine for 5 d, colon 26 cells (1 Â 104) were implanted into the spleens of BALB/c mice. A, representative pictures of the mouse livers (n = 5 for each group). a, 3 mg/d isoleucine; b, 0.6 mg/d isoleucine; c, 0.12 mg/d isoleucine; d, 3 mg/d isoleucine (nude mouse); e, 3 mg/d leucine. B, number of liver metastases 3 wks after treatment. *, P < 0.005; **, P < 0.01. C, liver weight 3 wks after treatment. *, P < 0.0001; **, P < 0.00002. D, serum albumin levels 3 wks after treatment.

Effect of isoleucine on VEGF-mediated angiogenesis in vitro. up-regulated in the cancerous lesions in the livers of the leucine- We next examined whether isoleucine affects angiogenesis, which treated mice (Fig. 4C). The expression levels of phosphorylated is an important process in tumor growth. In in vitro experiments, 4E-BP1 and Stat3 were parallel to the VEGF mRNA level. HIF1-a, isoleucine inhibited VEGF production by colon 26 cells in a however, was up-regulated in the isoleucine-treated mice relative dose-dependent manner (Fig. 3A), whereas leucine produced no to the leucine-treated mice, and quite low expression levels were effect on VEGF production relative to the medium alone. observed in the cancerous lesions. The expression level of HIF1-a Interestingly, a MTT assay showed that neither isoleucine nor was inversely correlated with the expression level of phosphor- leucine affected cell viability, even with a rather high concentration ylated 4E-BP1 and Stat3. In addition, immunohistochemical of isoleucine or leucine (1 mg/mL; Fig. 3B). The expression level staining of endothelial cells with anti-CD31 showed that the of VEGF mRNA in isoleucine-treated colon 26 cells was markedly number of endothelial cells in the tumor was markedly reduced lower than that in cells treated with leucine or medium alone in the isoleucine-treated mice (0.6 mg/d; Fig. 5B) relative to the (Fig. 3C and D). number in the leucine-treated mice (Fig. 5A). These results show Effect of isoleucine on angiogenesis in a mouse liver that isoleucine exerts antiangiogenic effects in a mouse liver metastatic model. To determine whether the antiangiogenic metastatic model as well as in in vitro cell cultures. effect of isoleucine could be seen in mice as well as in cell culture, VEGF mRNA levels were determined by RT-PCR using Discussion liver or spleen samples collected from mice treated with We show here that isoleucine prevents tumor growth in a isoleucine, leucine, or PBS. In nontumor lesions of the liver, the mouse liver metastatic model of colon cancer. Contrary to our expression level of VEGF mRNA in the isoleucine-treated mice was markedly impaired in comparison with the levels in the leucine-treated mice. Furthermore, the VEGF mRNA level in the leucine-treated mice was up-regulated relative to the expression levels in the PBS-treated mice (Fig. 4A and B). The VEGF mRNA level in the cancerous lesion was further up-regulated. The expression levels of VEGF mRNA in spleens treated with either isoleucine or leucine (without colon 26 challenge) tended to be higher than those in the livers of naive mice. Western blotting of nontumor liver tissues revealed that the expression of phosphorylated 4E-BP1 and Stat3 was down-regulated in the liver of the isoleucine-treated mice in comparison with the livers of the Figure 2. mBD-3 expression 3 wks after treatment with isoleucine (Iso; PBS-treated mice. Levels of these phosphorylated proteins were 3 mg/d) or leucine (Leu; 3 mg/d; RT-PCR). Representative data (n = 5 for each up-regulated in the leucine-treated mice, and they were further group). www.aacrjournals.org 3265 Cancer Res 2007; 67: (7). April 1, 2007

NKT cells. These observations imply that immunologic responses do not contribute to the mechanism of prevention of liver metastases by isoleucine. Next, we determined whether isoleucine has effects on angiogenesis because angiogenesis is generally an essential factor for the growth of various tumors (16–18). Our in vitro experiments showed that isoleucine inhibited VEGF production in a mouse colon cancer cell line at transcriptional levels. It is important that we did not use amino acid–deficient medium before cultivating of colon 26 cells with isoleucine or leucine, which should be similar condition that patients take additional isoleucine to daily food intake. Interestingly, isoleucine did not affect cell viability, implying that the inhibitory effect of isoleucine on VEGF production is not due to antiproliferative or direct effects on the VEGF-producing cells. These pieces of evidence were confirmed by an in vivo study, in which liver VEGF mRNA levels were found to be markedly reduced in the isoleucine-treated mice. The liver VEGF mRNA levels of the leucine-treated mice were up-regulated relative to the levels in the mice given PBS alone, and the VEGF mRNA levels were further up-regulated in the cancerous lesions of the leucine-treated

Figure 3. Effects of isoleucine and leucine on VEGF and VEGF mRNA production and cell viability in colon 26 cells. A, ELISA for VEGF in the culture medium showed that isoleucine inhibits VEGF production in a dose-dependent manner. B, MTT assays showed that neither isoleucine nor leucine exhibited any cytotoxicity. C and D, 2 d after seeding of colon 26 cells, the culture medium was replaced with isoleucine, leucine, or medium alone in 2.5% DMEM and cells were harvested after 24 h. Representative gels of RT-PCR for VEGF mRNA expression. predictions, this occurred because isoleucine inhibited VEGF, thus producing antiangiogenic effects, not because of tumor immunity via induction of h-defensin. Furthermore, isoleucine inhibited VEGF production partially through a mTOR pathway, not through a HIF1-a–dependent pathway. Importantly, isoleucine did not show any signs of cytotoxicity in our in vitro experiments. Figure 4. A and B, effects of isoleucine and leucine on VEGF mRNA levels in vivo. After they were pretreated with isoleucine or leucine (3 mg/d) for We initially hypothesized that isoleucine would prevent liver 5 d, colon 26 cells (1 Â 104) were implanted into the spleens of BALB/c mice. metastatic tumor growth via innate and acquired immunity, given Three weeks later, mice were sacrificed and their livers were removed for further that it is a strong inducer of h-defensins, which promote the studies. Representative gel from RT-PCR of VEGF mRNA (n = 5 each group) in mice liver. Lane 1, liver of isoleucine-treated mice (nontumor tissue); accumulation of memory T cells and dendritic cells through CCR6 lane 2, liver of leucine-treated mice (nontumor tissue); lane 3, liver metastatic (10). We found that isoleucine prevented liver metastasis in a lesion from leucine-treated mice; lane 4, liver from PBS-treated mice (without colon 26 challenge); lane 5, spleen from isoleucine-treated mice (without colon dose-dependent manner, but no mBD-3 expression and no 26 challenge); lane 6, spleen from leucine-treated mice (without colon 26 immunologic cytotoxicity was observed, even in the isoleucine- challenge). *, P < 0.05; **, P < 0.01. C, representative Western blot of treated mice, in which metastatic liver tumors were completely HIF1-a and phosphorylated 4E-BP1 and Stat3 proteins in mouse liver. Lane 1, isoleucine-treated mice (nontumor liver); lane 2, leucine-treated mice prevented. Furthermore, we found that isoleucine completely (nontumor liver); lane 3, cancer lesion from leucine-treated mouse; lane 4, prevented liver metastases in nude mice, which lack T cells and liver from PBS-treated mice (without colon 26 challenge).

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Figure 5. Liver tissue stained with anti-CD31. Representative liver metastatic lesions from mice treated with 3 mg/d leucine (A) or 0.6 mg/d isoleucine (B), n = 5 for each group. Bar,50Am.

mice. Furthermore, immunohistochemical staining of livers with regulates VEGF expression via a HIF1-a–independent pathway. anti-CD31 showed a marked reduction in the number of Many investigators have concluded that the phosphatidylinositol endothelial cells in the tumors of the isoleucine-treated mice 3-kinase/PTEN/Akt pathway can increase VEGF expression via (0.6 mg/d) in comparison with those of the leucine-treated mice. HIF1-a (28–30). However, Pore et al. (31) indicated that Akt can Collectively, these observations support the hypothesis that transactivate the VEGF promoter and lead to increased VEGF isoleucine prevents liver metastases through antiangiogenesis. expression-independent of HIF1-a. Their findings seem to support Interestingly, the levels of VEGF mRNA in the spleen were similar our findings. in both the isoleucine- and the leucine-treated mice. These results Leucine enhances albumin synthesis in rat hepatocytes, may indicate that, like leucine, isoleucine was not able to prevent whereas isoleucine does not (23). In the present study, the serum primary tumors in the spleen (the injection site). There are two albumin concentration of the leucine-treated mice was lower than explanations for these phenomena. The first explanation is that the that of the isoleucine-treated mice. This discrepancy may arise VEGF production is at a sufficiently high level in the naive spleen from the fact that the leucine-treated mice had multiple such that isoleucine cannot inhibit it because the spleen is a vessel- metastases in the liver, which may have impaired albumin rich organ. The second explanation is that the concentration of synthesis, whereas the isoleucine-treated mice did not show any isoleucine may be higher in the liver than that in the spleen liver metastases and looked healthy. Thus, albumin synthesis has because absorbed isoleucine from the intestine firstly passes been unaltered, although isoleucine itself is not able to enhance through the portal vein. protein synthesis. Clinically, treatment with branched-chain It has been reported recently that leucine promotes protein amino acids (BCAA) can actually improve serum albumin synthesis partially through the mTOR pathway (23, 24). Leucine concentrations in patients with liver cirrhosis. However, the enhances a signaling pathway that induces the protein kinase incidence of hepatocellular carcinoma seems to be similar mTOR, following induction of p70 S6 kinase activation and 4E-BP1 between patients with and without BCAA treatment. In cirrhotic phosphorylation, which are the downstream translational effectors patients treated with BCAA, leucine may enhance angiogenesis as of mTOR. These effects are blocked by rapamycin, which is a well as serum albumin concentrations partially through the specific inhibitor of mTOR (22). Oral administration of isoleucine mTOR pathway. At the same time, isoleucine may down-regulate as well as leucine promotes increased availability of eIF-4E, p70 S6 angiogenicity by impairment of VEGF production. These opposing kinase activation, and 4E-BP1 phosphorylation (23, 24). However, effects may counterbalance angiogenicity in cirrhotic patients these alterations by isoleucine in translation initiation do not lead being treated with BCAA. to a stimulation of protein synthesis (23, 24) or DNA synthesis (27). In conclusion, we found that isoleucine prevents tumor growth In both our in vivo and in vitro studies, isoleucine down-regulates in a mouse liver metastatic model. Furthermore, we determined VEGF mRNA expression and phosphorylation of 4E-BP1 and Stat3, that isoleucine prevents tumor growth via a novel mechanism, by which are the downstream transcriptional proteins of the mTOR impairment of VEGF production, partially through mTOR path- pathway. These results imply that isoleucine down-regulates VEGF way–independent HIF1-a. Importantly, isoleucine is only an through the mTOR pathway. These alterations of the mTOR essential amino acid, and no significant cytotoxicities against cells pathway are not sufficient to explain the down-regulation of VEGF were observed. Treatment with isoleucine could be a safe and in the present study, although these results are consistent with effective prophylactic treatment for use in patients with colon those of other studies (23, 24, 27). There may be other pathways or cancer, and clinical trials should be considered. mechanisms involved in the antiangiogenic effect of isoleucine. In the present study, the expression level of HIF1-a was inversely correlated with the expression level of phosphorylated 4E-BP1 and Acknowledgments Stat3. These findings suggest that isoleucine down-regulated the Received 10/16/2006; revised 12/31/2006; accepted 1/30/2007. transcriptional factor of VEGF through the mTOR pathway and The costs of publication of this article were defrayed in part by the payment of page that HIF1-a was up-regulated in a negative feedback process. charges. This article must therefore be hereby marked advertisement in accordance a with 18 U.S.C. Section 1734 solely to indicate this fact. Although the reason HIF1- was inversely up-regulated in our We thank M. Itoh and T. Nobori for helpful discussions and T. Inoue for technical experiment remains unclear, we have shown that isoleucine down- assistance. www.aacrjournals.org 3267 Cancer Res 2007; 67: (7). April 1, 2007

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis

Kazumoto Murata and Masami Moriyama

Cancer Res 2007;67:3263-3268.

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