Cancer Research Cancer Research Funding Available Protein Translation and 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
Cancer Research 67, 3263, April 1, 2007. doi: 10.1158/0008-5472.CAN-06-3739
© 2007 American Association for Cancer Research
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 Google Scholar
Google Scholar
Right arrow Articles by Murata, K.
Right arrow Articles by Moriyama, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murata, K.
Right arrow Articles by Moriyama, M.

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis

Kazumoto Murata1 and Masami Moriyama2

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

Requests for reprints: Kazumoto Murata, The First Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie, Japan 514-8507. Phone: 81-59-231-5015; Fax: 81-59-231-5201; E-mail: atarum{at}clin.medic.mie-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In spite of recent advances in the treatment of colon cancer, multiple liver metastases of colon cancer are still difficult to treat. Some chemotherapeutic regimens have been reported to be efficient, but there is a high risk of side effects associated with these. Here, we show that isoleucine, an essential amino acid, prevents liver metastases in a mouse colon cancer metastatic model. Because isoleucine is a strong inducer of ß-defensin, we first hypothesized that it prevented liver metastases via the accumulation of dendritic cells or memory T cells through up-regulation of ß-defensin. However, neither ß-defensin nor immunologic responses were induced by isoleucine in both mouse livers and spleens. Furthermore, isoleucine prevented liver metastasis in nude mice, which lack T cells and natural killer T cells. Finally, we discovered a novel mechanism of isoleucine: down-regulation of angiogenesis via inhibition of vascular endothelial growth factor, partially through the mammalian target of the rapamycin pathway, independent of hypoxia-inducible factor 1-{alpha}. Importantly, isoleucine is safe for administration to humans because it does not affect cell viability. Isoleucine could be a novel prophylactic drug for the prevention of liver metastases of colon cancer. [Cancer Res 2007;67(7):3263–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colorectal cancer is one of the leading causes of deaths from cancer worldwide. In Japan, the incidence of colorectal cancer has increased in association with diets becoming more "Westernized." In spite of recent advances in the treatment of colon cancer, the prognosis of patients with multiple liver metastases of colon cancers is still poor, and systemic chemotherapy prolongs survival only by a few months (1). Moreover, there is a high risk of severe side effects (2, 3), although methods for reducing the side effects of systemic chemotherapy [e.g., infusion chemotherapy through a reservoir pump (4) or chronotherapy (5)] can be used.

Antimicrobial peptides, such as defensins, play an important role in the innate immunity of all life forms, including plants or insects, which lack immune systems comprising such elements as antibodies or lymphocytes. Several types of antimicrobial peptides are induced at epithelial surfaces in response to inflammation. These antimicrobial peptides exhibit a broad spectrum of antifungal, antibacterial (6), and antiviral activities (7). Impairment of the function of defensin leads to susceptibility to infection in the airways of cystic fibrosis (8), and susceptibility to salmonella infection in mouse intestinal tracts (9). In addition to their direct antimicrobial activities, ß-defensins are strong chemotactic factors for memory T cells and dendritic cells, suggesting that they also play an important role in acquired immunity (10–12). ß-defensins are also inducible by inflammatory cytokines, such as tumor necrosis factor-{alpha} and interleukin-1ß (13, 14). Recently, Fehlbaum et al. (15) reported that isoleucine and its analogues are highly specific inducers of ß-defensins. We thus originally hypothesized that isoleucine may contribute to tumor immunity through both innate and acquired immunity by induction of ß-defensins.

Angiogenesis is a key process in tumor growth (16–18), and vascular endothelial growth factor (VEGF), which stimulates angiogenesis in a paracrine fashion, seems to be essential for the progression of various solid tumors. VEGF production is initiated by phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) by the mammalian target of the rapamycin (mTOR) following disruption of their binding to eIf-4E, enabling eIF-4E to translate most mRNAs, including VEGF, through the m7GpppN cap (19–21). Rapamycin, an immunosuppressive drug, interacts with mTOR, following dephosphorylation of 4E-BP1 and 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 eIF-4E (23, 24). However, the association between isoleucine and angiogenesis remains unclear.

Here, we describe remarkable results indicating that isoleucine actually prevents tumor growth in a mouse colon cancer liver metastatic model. The mechanism by which isoleucine exerts its antitumor effect was not, as we expected, via an immunologic pathway through the up-regulation of ß-defensins. Rather, isoleucine acts through a novel mechanism whereby it inhibits VEGF at the translation initiation level, partially through the mTOR pathway. Importantly, isoleucine did not affect cell viability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell lines. Male BALB/c mice (6–8 weeks old) and male BALB/c nude mice (nu/nu, 6–8 weeks old) were obtained from SLC (Shizuoka, Japan) and maintained with ad libitum access to food and water. Colon 26 cells (BALB/c syngenic colon cancer cells) were maintained in DMEM (Sigma, Tokyo, Japan) containing 10% FCS. Yac-1 cells 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 feeding tube, starting from 5 days before the tumor implantation until sacrifice. On the operation day, 1 x 104 colon 26 cells in 0.2 mL PBS were implanted into the spleens of anesthetized and laparotomized mice. Three weeks after the operation, the mice were sacrificed and examined for liver metastases. Blood samples were taken by retro-orbital puncture and serum albumin concentrations were measured using a standard clinical automatic analyzer. The liver of each mouse was weighed. Livers, spleens, and tracheas were removed and frozen until they were used for reverse transcription-PCR (RT-PCR) and Western blotting studies.

Immunologic experiments. Seven days after administration of isoleucine or leucine (3 mg/d; n = 5 each), mice were sacrificed and intrahepatic lymphocytes were prepared as reported previously (25). The intrahepatic lymphocytes and splenic lymphocytes were stained with anti-CD3 and anti-panNK (anti-DX5; BD Biosciences, San Jose, CA). The intrahepatic lymphocytes and splenic lymphocytes were subjected to a standard natural killer (NK) cytotoxic assay. Briefly, various numbers of intrahepatic lymphocytes and splenic lymphocytes were cultured with 3,000 51Cr-coated Yac-1 cells for 5 h. The amount of 51Cr in the supernatant was measured. The CTL concentration was measured using a standard 5-h chromium releasing assay. Briefly, splenocytes (nontumor site) from the isoleucine-treated mice (3 mg/mL; n = 5) 3 weeks after implantation of colon 26 cells or naive mice were incubated with irradiated (10,000 rad) colon 26 cells in complete culture medium with 10% T-stim (Becton Dickinson, Billerica, MA) for 7 days following coculture with chromium-coated colon 26 cells for 5 h. The amount of chromium released into the medium was measured. Specific killing was calculated using the following equation: (experimental release – spontaneous release) / (maximum release – spontaneous release) x 100.

In vitro experiments. Two days after seeding of colon 26 cells at a cell density of 1 x 104 cells/cm2 to a 24-well plate, the medium was replaced with isoleucine or leucine dissolved in 2.5% FCS containing DMEM, and the cells were cultured for another 48 h. The supernatant was used to measure the VEGF concentration by ELISA (mouse VEGF ELISA kit; R&D Systems, Minneapolis, MN). These cells were used for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well for checking for any effects of the amino acids on cell viability. For RT-PCR, cells were harvested 24 h after replacement of the medium.

RT-PCR. RT-PCR was used to measure the concentration of mouse ß-defensin (mBD)-3 and VEGF in the mouse tissues or in the colon 26 cells. Poly(A)+RNA was isolated using RNA Zol (Biotecx Laboratories, Houston, TX) from mouse tissues (liver, spleen, and trachea) or cells and a 1 µg aliquot was reverse transcribed into cDNA (Taqman; Applied Biosystems, Foster City, CA). The following primers were used for PCR: BD-3, 5'-CTCTTTGCATTTCTCCTGGTGCTGCTG-3' (forward) and 5'-CATCTTCATGGAGGAGCAAATTCTG-3' (reverse); VEGF, 5'-GCGGGCTGCCTCGCAGTC-3' (forward) and 5'-TCACCGCCTTGGCTTGTAC-3' (reverse). RT-PCR using primers specific for mouse glyceraldehyde-3-phosphate dehydrogenase was used as a positive control. Trachea tissue was used as a positive control for mBD-3 mRNA.

Western blotting. The cell extracts were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking, the membranes were incubated overnight at 4°C with antibodies against hypoxia-inducible factor 1-{alpha} (HIF1-{alpha}; Santa Cruz Biotechnology, Santa Cruz, CA) or phosphospecific antibodies against 4E-BP1 (Thr37/46; Cell Signaling Technology, San Diego, CA) and signal transducers and activators of transcription 3 (Stat3; Santa Cruz Biotechnology). Antibodies against {alpha}-tubulin were used as an internal control. The membranes were washed and then incubated for 30 min with horseradish peroxidase–conjugated secondary antibodies. The membranes were washed again, and the proteins were detected using electrochemiluminescence techniques (Pierce Chemicals, Rockford, IL).

Immunohistochemical staining for CD31. Immunohistochemistry was done using a peroxidase-streptavidin method on frozen livers, using antibodies against mouse CD31 (BD PharMingen, San Diego, CA). The antibodies were detected with 3,3'-diaminobenzidine. The sections were counterstained with 10% hematoxylin (Wako Pure Chemical, Osaka, Japan).

Statistical analysis. Values are expressed as means ± SD. Multiple comparisons were done using one-way ANOVA. Intergroup comparisons were done using Bonferroni's correction for multiple comparisons. A level of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of isoleucine on liver metastasis of colon cancer. We first hypothesized that because isoleucine induces ß-defensins, it could contribute to tumor regression through innate or acquired immunity. Thus, we attempted to determine whether isoleucine prevents tumor growth in a mouse liver metastatic model of colon cancer. Remarkably, none of the isoleucine-treated mice (3 mg/d) had any liver metastases (n = 5). In contrast, no tumor growth inhibition was observed in the leucine-treated mice (n = 5), relative to mice treated with PBS (data not shown). These effects were observed with a rather low concentration of isoleucine (0.6 mg/d), and the effect on the number of liver metastases was dose dependent (Fig. 1A and B ). The effects of isoleucine against liver metastases were supported by observations with respect to both liver weight (Fig. 1C) and serum albumin levels (Fig. 1D). Interestingly, tumor cells in the spleen were found to be growing even in the isoleucine-treated mice (3 mg/d), which did not have any liver metastases. Furthermore, the sizes of the tumors in the spleen in the isoleucine-treated mice were similar to the sizes of those in the leucine-treated mice (data not shown).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		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 days, colon 26 cells (1 x 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.

 
Isoleucine does not induce mBD-3 in mice. Because isoleucine is a strong inducer of ß-defensins (15), we initially hypothesized that ß-defensins would be induced in the liver by isoleucine treatment. Of the many ß-defensins, we focused on mBD-3 because human ß-defensin-2, which is the homologue of mouse BD-3, causes the accumulation of dendritic cells or memory T cells around tumors and contributes to tumor immunity (10). Additionally, mBD-2 and mBD-3 are chemotactics for immature dendritic cells (12) and mBD-3 is inducible in the liver (26). Therefore, using RT-PCR, we examined mBD-3 mRNA expression in the livers and the spleens of mice treated with isoleucine or leucine. However, no expression of mBD-3 was observed in either liver or spleen tissue, even in mice, in which liver metastases were prevented by isoleucine treatment, whereas mBD-3 mRNA was clearly detected in trachea tissue (positive control; Fig. 2 ). Thus, it seems that the expression of mBD-3 does not contribute to the prevention of liver metastases by isoleucine treatment in our model.


Figure 2
View larger version (9K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. mBD-3 expression 3 wks after treatment with isoleucine (Iso; 3 mg/d) or leucine (Leu; 3 mg/d; RT-PCR). Representative data (n = 5 for each group).

 
Isoleucine does not induce innate or acquired immunity in liver or spleen. To investigate the immunologic processes participating in the prevention of liver metastases of colon cancer by isoleucine, NK cytotoxicity was examined. NK cytotoxicity was not induced in either the intrahepatic lymphocytes (3.8 ± 2.1%; E:T, 50:1) or the splenocytes (6.9 ± 5.1%; E:T, 50:1) of the isoleucine-treated mice, implying that neither NK nor NKT cells have an important role in the prevention of liver metastases (naive mice; intrahepatic lymphocytes, 5.2 ± 0.3%; splenocytes, 8.3 ± 1.8%, respectively). Furthermore, no significant differences in the populations of intrahepatic lymphocytes or splenocytes were observed between the isoleucine-treated mice and naive mice (data not shown). A CTL assay using splenocytes from the isoleucine-treated mice without any liver metastases showed that no specific killing of colon 26 cells was occurring (isoleucine, 5.9 ± 4.9%; leucine, 7.0 ± 2.1%, respectively; E:T, 50:1). Furthermore, isoleucine (3 mg/d) completely prevented liver metastases even in nude mice (n = 5), implying that neither T cells nor NKT cells have an important role in the prevention of liver metastases by isoleucine (Fig. 1). Taken together, these results show that neither innate nor acquired immunity plays a major role in the prevention of metastatic liver tumors by isoleucine.

Effect of isoleucine on VEGF-mediated angiogenesis in vitro. We next examined whether isoleucine affects angiogenesis, which is an important process in tumor growth. In in vitro experiments, isoleucine inhibited VEGF production by colon 26 cells in a dose-dependent manner (Fig. 3A ), whereas leucine produced no effect on VEGF production relative to the medium alone. Interestingly, a MTT assay showed that neither isoleucine nor leucine affected cell viability, even with a rather high concentration of isoleucine or leucine (1 mg/mL; Fig. 3B). The expression level of VEGF mRNA in isoleucine-treated colon 26 cells was markedly lower than that in cells treated with leucine or medium alone (Fig. 3C and D).


Figure 3
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		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.

 
Effect of isoleucine on angiogenesis in a mouse liver metastatic model. To determine whether the antiangiogenic effect of isoleucine could be seen in mice as well as in cell culture, VEGF mRNA levels were determined by RT-PCR using liver or spleen samples collected from mice treated with isoleucine, leucine, or PBS. In nontumor lesions of the liver, the 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 PBS-treated mice. Levels of these phosphorylated proteins were up-regulated in the leucine-treated mice, and they were further up-regulated in the cancerous lesions in the livers of the leucine-treated mice (Fig. 4C). The expression levels of phosphorylated 4E-BP1 and Stat3 were parallel to the VEGF mRNA level. HIF1-{alpha}, however, was up-regulated in the isoleucine-treated mice relative to the leucine-treated mice, and quite low expression levels were observed in the cancerous lesions. The expression level of HIF1-{alpha} was inversely correlated with the expression level of phosphorylated 4E-BP1 and Stat3. In addition, immunohistochemical staining of endothelial cells with anti-CD31 showed that the number of endothelial cells in the tumor was markedly reduced in the isoleucine-treated mice (0.6 mg/d; Fig. 5B ) relative to the number in the leucine-treated mice (Fig. 5A). These results show that isoleucine exerts antiangiogenic effects in a mouse liver metastatic model as well as in in vitro cell cultures.


Figure 4
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		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 5 d, colon 26 cells (1 x 104) were implanted into the spleens of BALB/c mice. Three weeks later, mice were sacrificed and their livers were removed for further 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); lane 2, liver of leucine-treated mice (nontumor tissue); lane 3, liver metastatic 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 26 challenge); lane 6, spleen from leucine-treated mice (without colon 26 challenge). *, P < 0.05; **, P < 0.01. C, representative Western blot of HIF1-{alpha} and phosphorylated 4E-BP1 and Stat3 proteins in mouse liver. Lane 1, isoleucine-treated mice (nontumor liver); lane 2, leucine-treated mice (nontumor liver); lane 3, cancer lesion from leucine-treated mouse; lane 4, liver from PBS-treated mice (without colon 26 challenge).

 

Figure 5
View larger version (73K):
[in this window]
[in a new window]
[Download PPT slide]
  		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, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show here that isoleucine prevents tumor growth in a mouse liver metastatic model of colon cancer. Contrary to our predictions, this occurred because isoleucine inhibited VEGF, thus producing antiangiogenic effects, not because of tumor immunity via induction of ß-defensin. Furthermore, isoleucine inhibited VEGF production partially through a mTOR pathway, not through a HIF1-{alpha}–dependent pathway. Importantly, isoleucine did not show any signs of cytotoxicity in our in vitro experiments.

We initially hypothesized that isoleucine would prevent liver metastatic tumor growth via innate and acquired immunity, given that it is a strong inducer of ß-defensins, which promote the accumulation of memory T cells and dendritic cells through CCR6 (10). We found that isoleucine prevented liver metastasis in a dose-dependent manner, but no mBD-3 expression and no immunologic cytotoxicity was observed, even in the isoleucine-treated mice, in which metastatic liver tumors were completely prevented. Furthermore, we found that isoleucine completely prevented liver metastases in nude mice, which lack T cells and 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 mice. Furthermore, immunohistochemical staining of livers with anti-CD31 showed a marked reduction in the number of endothelial cells in the tumors of the isoleucine-treated mice (0.6 mg/d) in comparison with those of the leucine-treated mice. Collectively, these observations support the hypothesis that isoleucine prevents liver metastases through antiangiogenesis. Interestingly, the levels of VEGF mRNA in the spleen were similar in both the isoleucine- and the leucine-treated mice. These results may indicate that, like leucine, isoleucine was not able to prevent primary tumors in the spleen (the injection site). There are two explanations for these phenomena. The first explanation is that the VEGF production is at a sufficiently high level in the naive spleen such that isoleucine cannot inhibit it because the spleen is a vessel-rich organ. The second explanation is that the concentration of isoleucine may be higher in the liver than that in the spleen because absorbed isoleucine from the intestine firstly passes through the portal vein.

It has been reported recently that leucine promotes protein synthesis partially through the mTOR pathway (23, 24). Leucine enhances a signaling pathway that induces the protein kinase mTOR, following induction of p70 S6 kinase activation and 4E-BP1 phosphorylation, which are the downstream translational effectors of mTOR. These effects are blocked by rapamycin, which is a specific inhibitor of mTOR (22). Oral administration of isoleucine as well as leucine promotes increased availability of eIF-4E, p70 S6 kinase activation, and 4E-BP1 phosphorylation (23, 24). However, these alterations by isoleucine in translation initiation do not lead to a stimulation of protein synthesis (23, 24) or DNA synthesis (27). In both our in vivo and in vitro studies, isoleucine down-regulates VEGF mRNA expression and phosphorylation of 4E-BP1 and Stat3, which are the downstream transcriptional proteins of the mTOR pathway. These results imply that isoleucine down-regulates VEGF through the mTOR pathway. These alterations of the mTOR pathway are not sufficient to explain the down-regulation of VEGF in the present study, although these results are consistent with those of other studies (23, 24, 27). There may be other pathways or mechanisms involved in the antiangiogenic effect of isoleucine. In the present study, the expression level of HIF1-{alpha} was inversely correlated with the expression level of phosphorylated 4E-BP1 and Stat3. These findings suggest that isoleucine down-regulated the transcriptional factor of VEGF through the mTOR pathway and that HIF1-{alpha} was up-regulated in a negative feedback process. Although the reason HIF1-{alpha} was inversely up-regulated in our experiment remains unclear, we have shown that isoleucine down-regulates VEGF expression via a HIF1-{alpha}–independent pathway. Many investigators have concluded that the phosphatidylinositol 3-kinase/PTEN/Akt pathway can increase VEGF expression via HIF1-{alpha} (28–30). However, Pore et al. (31) indicated that Akt can transactivate the VEGF promoter and lead to increased VEGF expression-independent of HIF1-{alpha}. Their findings seem to support our findings.

Leucine enhances albumin synthesis in rat hepatocytes, whereas isoleucine does not (23). In the present study, the serum albumin concentration of the leucine-treated mice was lower than that of the isoleucine-treated mice. This discrepancy may arise from the fact that the leucine-treated mice had multiple metastases in the liver, which may have impaired albumin synthesis, whereas the isoleucine-treated mice did not show any liver metastases and looked healthy. Thus, albumin synthesis has been unaltered, although isoleucine itself is not able to enhance protein synthesis. Clinically, treatment with branched-chain amino acids (BCAA) can actually improve serum albumin concentrations in patients with liver cirrhosis. However, the incidence of hepatocellular carcinoma seems to be similar between patients with and without BCAA treatment. In cirrhotic patients treated with BCAA, leucine may enhance angiogenesis as well as serum albumin concentrations partially through the mTOR pathway. At the same time, isoleucine may down-regulate angiogenicity by impairment of VEGF production. These opposing effects may counterbalance angiogenicity in cirrhotic patients being treated with BCAA.

In conclusion, we found that isoleucine prevents tumor growth in a mouse liver metastatic model. Furthermore, we determined that isoleucine prevents tumor growth via a novel mechanism, by impairment of VEGF production, partially through mTOR pathway–independent HIF1-{alpha}. Importantly, isoleucine is only an essential amino acid, and no significant cytotoxicities against cells were observed. Treatment with isoleucine could be a safe and effective prophylactic treatment for use in patients with colon cancer, and clinical trials should be considered.


   Acknowledgments
 
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 M. Itoh and T. Nobori for helpful discussions and T. Inoue for technical assistance.

Received 10/16/06. Revised 12/31/06. Accepted 1/30/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Simmonds PC; Colorectal Cancer Collaborative Group. Palliative chemotherapy for advanced colorectal cancer: systemic review and meta-analysis. BMJ 2000;321:531–5.[Abstract/Free Full Text]
  2. Sargent DJ, Niedzwiecki D, O'Connell MJ, Schilsky RL. Recommendation for caution with irinotecan, fluorouracil, and leucovorin for colorectal cancer. N Eng J Med 2001;345:144–5.[Free Full Text]
  3. Delaunoit T, Goldberg RM, Sargent DJ, et al. Mortality associated with daily bolus 5-fluorouracil/leucovorin administered in combination with either irinotecan or oxaliplatin: results from Intergroup Trial N9741. Cancer 2004;101:2170–6.[CrossRef][Medline]
  4. de Gramont A, Bosset JF, Milan C, et al. Randomized trial comparing monthly low-dose leucovorin and fluorouracil bolus with bimonthly high-dose leucovorin and fluorouracil bolus plus continuous infusion for advanced colorectal cancer: a French intergroup study. J Clin Oncol 1997;15:808–15.[Abstract/Free Full Text]
  5. Levi F, Zidani R, Misset JL; International Organization for Cancer Chronotherapy. Randomized multicentre trial of chronotherapy with oxliplatin, fluorouracil, and folinic acid in metastatic colorectal cancer. Lancet 1997;350:681–6.[CrossRef][Medline]
  6. Schonwetter BS, Stolzenberg ED, Zasloff MA. Epithelial antibiotics induced at sites of inflammation. Science 1995;267:1645–8.[Abstract/Free Full Text]
  7. Daher KA, Selsted ME, Lehrer RI. Direct inactivation of viruses by human granulocyte defensins. J Virol 1986;60:1068–74.[Abstract/Free Full Text]
  8. Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilsor JM. Human ß-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997;21:553–60.
  9. Wilson CL, Ouellette AJ, Satchell DP, et al. Regulation of intestinal {alpha}-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 1999;286:113–7.[Abstract/Free Full Text]
  10. Yang D, Chertov O, Bykovskaia SN, et al. ß-Defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999;286:525–8.[Abstract/Free Full Text]
  11. Chertov O, Yang D, Howard OMZ, Oppenheim JJ. Leukocyte granule proteins mobilize innate host defenses and adaptive immune responses. Immunol Rev 2000;177:68–78.[CrossRef][Medline]
  12. Biragyn A, Surenhu M, Yang D, et al. Mediators of innate that target immature, but mature, dendritic cells induce antitumor immunity when generally fused with nonimmunogenic tumor antigen. J Immunol 2001;167:6644–53.[Abstract/Free Full Text]
  13. Russell JP, Diamond G, Tarver AP, Scanlin TF, Bevins CL. Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor {alpha}. Infect Immun 1996;64:1565–8.[Abstract]
  14. Mathews M, Jia HP, Guthmiller JM, et al. Production of ß-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect Immun 1999;67:2740–5.[Abstract/Free Full Text]
  15. Fehlbaum P, Rao M, Zasloff M, Anderson GM. An essential amino acid induces epithelial ß-defensin expression. Proc Natl Acad Sci U S A 2000;7:12723–8.
  16. Folkman J. Tumor angiogenesis: therapeutic implications. N Eng J Med 1971;285:1182–6.[Medline]
  17. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4–6.[Free Full Text]
  18. Holmgren L, O'Reilly MS, Folkman J. Dormacy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995;1:149–53.[CrossRef][Medline]
  19. De Benedetti A, Harris AL. eIF-4E expression in tumors: its possible role in progression of malignancies. Int J Biochem Cell Biol 1999;31:59–72.[CrossRef][Medline]
  20. Raught B, Gingras AC. eIF4E activity is regulated at multiple levels. Int J Biochem Cell Biol 1999;31:43–57.[CrossRef][Medline]
  21. Chung J, Bachelder RE, Lipscomb EA, Shaw LM, Mercurio AM. Integrin ({alpha}6ß4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells. J Cell Biol 2002;158:165–74.[Abstract/Free Full Text]
  22. Guba M, Breitenbuch PV, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002;8:128–35.[CrossRef][Medline]
  23. Anthony JC, Yoshizawa F, Anthony TG, Vary TC, Jefferson LS, Kimball SR. Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J Nutr 2000;130:2413–9.[Abstract/Free Full Text]
  24. Ijichi C, Matsumura T, Tsuji T, Eto Y. Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system. Biochem Biophys Res Commun 2003;303:59–64.[CrossRef][Medline]
  25. Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 2000;192:921–30.[Abstract/Free Full Text]
  26. Bals R, Wang X, Meegalla RL, et al. Mouse ß-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs. Infect Immun 1999;67:3542–7.[Abstract/Free Full Text]
  27. Kimura M, Ogihara M. Effects of branched-chain amino acids on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Eur J Pharmacol 2005;510:167–80.[CrossRef][Medline]
  28. Jiang BH, Zheng JZ, Aoki M, Vogt PK. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci U S A 2000;97:1749–53.[Abstract/Free Full Text]
  29. Zhong H, Chiles K, Feldser D, et al. Modulation of hypoxia-inducible factor 1{alpha} expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 2000;60:1541–5.[Abstract/Free Full Text]
  30. Zundel W, et al. Loss of PTEN facilities HIF-1-mediated gene expression. Genes Dev 2000;14:391–6.[Abstract/Free Full Text]
  31. Pore N, Liu S, Shu HK, et al. Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism. Mol Biol Cell 2004;15:4841–53.[Abstract/Free Full Text]




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 Google Scholar
Google Scholar
Right arrow Articles by Murata, K.
Right arrow Articles by Moriyama, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murata, K.
Right arrow Articles by Moriyama, M.

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