The goal of this study was to examine metabolic therapies using the newly established VM-M3 mouse model for metastatic cancer. We found that the VM-M3 tumor cell line was more dependent on glutamine than glucose for survival in vitro
. In support of our in vitro
data we showed that the glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), had a significant inhibitory effect on VM-M3 growth in vitro
and on tumor growth and metastasis in vivo
. In contrast, calorie restriction, which lowers circulating glucose levels, did not significantly reduce metastasis. This in vivo
finding supports the in vitro
findings that the VM-M3 tumor cells depend more on glutamine than on glucose for survival. As long as glutamine is available, the VM-M3 cells can survive despite the targeting of glucose. We previously showed that CR is anti-angiogenic and anti-inflammatory against experimental brain tumors 8, 9
. The anti-angiogenic and anti-inflammatory action of CR could be responsible for the reduction in primary tumor size observed in this study. However, evidence shows that circulating glutamine levels are increased in mice during calorie restriction 45
. In addition, glutamine levels are normally higher than that of any other circulating amino acid 46–48
. As glutamine promotes VM-M3 cell growth and survival, the failure of CR to target glutamine levels could be responsible for the failure of CR to inhibit VM-M3 tumor metastasis. Survival in CR mice was significantly longer than control mice in spite of the presence of systemic metastasis. This suggests that morbidity in the control mice may be due to a combination of factors to include primary tumor burden. As shown, the primary tumor size of mice on CR was approximately half the size of control mice. As previously shown, drugs, such as methotrexate, that inhibit metastasis but do not affect primary tumor size do not increase mouse survival 3
. Therefore, at the time of control mouse morbidity, systemic metastasis may not be a major contributing factor.
Increasing evidence indicates that glutamine is a major energy substrate for cancer cells, to include glioma, HeLa, and prostate cancer cell lines 12–15
. In addition, it is well known that glutamine is a major energy substrate for cells of myeloid origin 42
. Interestingly, the VM-M3 cell line has a number of properties in common with cells of myeloid/mesenchymal lineage (macrophages and microglia) to include morphology, gene expression, lipid profile, and phagocytic capacity 36
. We also found that the VM-M3 tumor cells require glutamine as a major energy substrate similar to cell of myeloid origin. Therefore, we suggest that the dependence of the VM-M3 cells on glutamine results from the myeloid origin of the cells.
We showed that DON administration in vitro
inhibited cell growth, but did not significantly enhance cell death. DON administration in vitro
therefore displayed a cytostatic effect over a wide range of concentrations, similar to other known cytostatic agents 3
. Additionally, whole body bioluminescence was nearly unchanged over the course of DON treatment with the exception of spleen metastasis (data not shown). These findings suggest that DON inhibits cell growth, consistent with previous reports in human studies 28
. There have also been reports of tumor regression, indicating that DON can be useful for early and late stage tumor management 17, 28
Although DON inhibited VM-M3 metastasis to the liver, lung, and kidneys, DON treatment had no effect on metastasis to the spleen. The spleen is recognized as a reservoir for monocytes 49
, and may represent a sanctuary for the myeloid-like metastatic cells. Interestingly, studies have shown increases in glutaminase activity in the spleens of tumor bearing mice 50
. Glutaminase is the first enzyme involved in glutamine metabolism. This perhaps indicates that the spleen could support tumor growth due to an influx of glutamine originally intended to support immune function 51
. Further studies are required to determine the factors involved in tumor cell metastasis to the spleen.
Because targeting glucose and glutamine individually increased mouse survival, we suggest that targeting both glucose and glutamine in vivo
could potentially have a synergistic and less toxic effect 52
. We previously found that CR administered together with low dose 2-deoxyglucose, an inhibitor of glucose metabolism, acted synergistically to reduce brain tumor growth 10
. In addition, DON treatment, in combination with 2-deoxyglucose, had a greater inhibitory effect on myeloid leukaemia cells in vitro25
. Previous studies suggest that glutamine inhibition also restricts glucose metabolism 53
. This suggests that some tumor cells might become more susceptible to glycolysis inhibition following inhibition of glutamine metabolism. Additionally, diets low in glutamate may further inhibit tumor growth and metastasis when combined with glutamine antagonists 54
. Further studies are required to evaluate the therapeutic efficacy of targeting both glutamine and glucose metabolism for the management of metastatic cancers.
Besides DON, phenylbutyrate (PBA), which reduces circulating glutamine levels, has been evaluated in human clinical trials 19–22, 27
. PBA is metabolized to phenylacetate (PA) in humans, which then covalently conjugates with glutamine18, 55
. This PA-glutamine conjugate is then excreted, effectively reducing circulating glutamine levels 18
. Although current studies utilize PBA as a histone deacetylase inhibitor in vitro
, part of its mechanism of action could be due to a reduction of circulating glutamine in vivo
. We were unable to test this possibility in mice, as PBA is metabolized differently in mice than in humans. PA is conjugated to glycine rather than glutamine in mice 56
. We therefore tested DON because of its previous use in mice 26
. Because PBA has already been introduced in clinical trials and is well tolerated in humans 55
, PBA can potentially be used in place of DON as a glutamine-targeting drug. Because PBA is well tolerated, the toxicities seen with DON treatment could be avoided. We suggest that new non-toxic inhibitors of glutamine metabolism could be broadly effective for managing systemic metastatic cancer.
In contrast to our data, previous studies showed that glutamine inhibited the formation of chemically induced squamous cell cancer 57
. However, cancers of epithelial origin may have different requirements for glutamine than cells of myeloid origin. In fact, we have shown that the CT2A astrocytoma, which is of epithelial origin, is highly dependent on glucose and is responsive to CR 9
. In addition, glutamine administration would be expected to enhance the activity of host immune cells such as macrophages. Macrophage function is highly dependent on glutamine 42
. In contrast, some reports suggest that glutamine supplementation can inhibit tumor growth 58
. Such inhibition was due to reductions in glutathione synthesis, which would render the tumor cells more susceptible to oxidative stress 58
. However, our unpublished findings, using C13
glutamine, show that the VM-M3 cells actively synthesize glutathione. Hence, glutamine is a necessary nutrient for the VM-M3 metastatic cells and possibly for human metastatic cells with myeloid/mesenchymal properties.
In conclusion, we found that glutamine is a major energy metabolite for the metastatic VM-M3 cells and suggest that targeting glutamine could be effective for managing systemic metastatic cancer in humans. We suggest that glutamine-targeting drugs could be more therapeutic and possibly less toxic than other current therapies for cancer metastasis. As glucose and glutamine are the primary energy metabolites of most malignant cancers, therapeutic synergy can be expected if these metabolites are targeted simultaneously.