Previous studies have shown that a lack of sufficient amount of glucose causes apoptosis in MYC transduced cells. This has not been shown to be the case in parental cell lines, only in MYC cells. This paper, however proposes an opposing theory. The article suggests that the mechanism of cell death via glucose depletion is not by apoptosis. Instead, it is proposed that MYC-dependent apoptosis is actually enhanced with the lack of adequate amounts of glutamine and not glucose deprivation when converting to ATP as previous studies suggested. This leads us up to the current paper where the metabolism of glucose is researched to observe the effects it may have on cell viability and consequently its role in cancer.
Info from Introduction
Since tumor cells are thought to uptake glucose and glutamine primarily, glucose and glutamine metabolism have long since been the main nutrients studied when researching tumor cells and cancer therapy. There have been many studies that have supported the idea that glucose (and its conversion to ATP) is a key player in growth and viability of the cells, and consequently apoptosis.
Articles, such as the Zu and Guppy article refute the idea that glucose depletion is a critical factor in the production of ATP and cell death via apoptosis. This article suggests that in spite of a lack of glucose, ATP can still be made via oxidative phosphorylation in both cancer and normal cells. It also suggests in this paper that it is possible for these tumor cells to survive if glutamine is provided even in the absence of glucose. Evidence that suggests that glutamine plays a role in tumor cell growth is the correlation between the tumor growth rate and the glutamine metabolism activity. Which leads us to the current paper which will show that glutamine is more important that glucose when it comes to cancer cells and cancer therapy.
Although it also recognized as being a nutrient that cells uptake, glutamine has not gotten the attention that glucose has in cancer therapy. Glutamine is shown to serve many functions in the body. One major function is producing ATP via the Citric acid cycle. This role in ATP is one that would suggest that glutamine plays a critical role in cell growth, survival and apoptosis.
The study discussed in this paper looked for large differences between MYC cells and normal cells. This study uses human cells. A quick summary of the results show that glucose depletion does not equate to apoptosis but, conversely, glutamine depletion does in fact induce apoptosis. The overall purpose of this study is determine both the roles of glutamine and glucose in normal and cancer cells in order to gain further insight on how to provide cell therapy. The article will often refer to ER-MYC and OHT. ER-MYC is a proto-oncogene (MYC) with an estrogen receptor binding domain (ER). The OHT binds ER-MYC and activates it.
Info from Results
Figure 1 observes the effect that the MYC activation has on the cells with glucose and with the depletion of glucose in human fibroblasts. The researchers wanted to determine whether glucose depletion is dependent or independent of the activation of MYC. Annexin V is a stain used to detect cells that undergo cell death. As seen in figure 1A, the percentage of cells that showed the staining with activated MYC and glucose present (MYCON â€" glucose) were similar to the inactivated MYC cells with glucose present. Figure 1A also shows that even with the depletion of glucose, the activated and inactivated MYC cells show annexin V staining percentages that are nearly identical. Figure A shows the cells over a 24 hour time span. Figure B, on the other hand, provides the same experiment at t=12hours and t=18hours. The findings here support the findings in part A. The cells show the same amount of annexin staining regardless of whether OHT was present (MYCON) or not (MYCOFF). Figure C shows fluorescent microscopy observation of ER-MYC cells with (+) and without (-) glucose. In part D, Bcl-2 is introduced into the cells. Bcl-2 is known to inhibit cell death via apoptosis. Figure D does not show the introduction of Bcl-2 into the MYC cells as inhibiting cell death, as is apparent by comparing to the vector cells.
The common finding that figures 1A-1D show is that the effects of glucose depletion in the cells is not dependent upon OHT and the activation of MYC. The lack of variation of annexin staining in part 1 and the lack of cell death inhibition by Bcl-2 in part D suggests that apoptosis is not the mechanism by which glucose affects cell death. An apoptotic mechanism of cell death would be expected to show Bcl-2 inhibiting cell death, which is not the case here.
Figure 2 makes observations on effects of glutamine deprivation in OHT activated ER-MYC cells. Figure 2A shows that cell death occurred significantly faster with activated MYC cells than with inactivated MYC cells when there is a lack of sufficient glutamine. This is different than what was observed when there was glucose depletion, where the percentage off annexin staining showed no difference between activated and inactivated MYC cells. In figure 2B, glutamine (-) cells show more condensing of the chromatin than glutamine (+) cells, which is a sign of apoptosis. Figure 2C shows another graphical representation of the effects that OHT activation of MYC has on cell death. Cells with glutamine showed very little amount of condensed chromatin which confirms the findings in 2B. With a lack of glutamine, activation of MYC greatly increases the percentage of cells with condensed chromatin. This confirms the findings in the previous two parts of figure 2.
Figure 3 shows the results of a test to see whether glutamine depletion induced apoptosis via intrinsic or extrinsic pathway. In order to do this, they transduced ER-MYC cells with inhibitors of the intrinsic and extrinsic pathways. ER-MYC cells were transduced with Bcl-2 with and without OHT present to activate the MYC cells and incubated. They also incubated vector cells as well. The vectors showed results consistent with those in figure 2, depleting glucose and the presence of OHT increases cell the percentage of cells stained with annexin. The cells transduced with Bcl-2 showed different results, however. The presence of OHT in glucose depleted cells transduced with Bcl-2 did not show an increase in percentage of annexin staining. This suggests that Bcl-2 is inhibiting apoptosis, even in the presence of OHT and activated ER-MYC cells. The experiment if figure 3B was conducted just as the one in 3A except that Caspase-9DN was transduced into the cells. The results with the caspase-9DN transduction were very similar to that of the Bcl-2. In the third experiment in figure 3C, cells were observed with alpha-CD95 and CrmA transduced into the cells. The vector cells were similar to those previously stated in figures 3a and 3b. The glaring difference is shown in the crmA transduced cells. Unlike the Bcl-2 and caspase-9DN cells, the crmA transduction did not inhibit cell death in the presence of OHT and with glucose depletion. Since both Bcl-2 and caspase-9DN are intrinsic pathway inhibitors and crmA is an extrinsic pathway inhibitor, they came to the conclusion that apoptosis was induced via the intrinsic pathway.
It still is not fully understood how or why glutamine affects apoptosis in cells. It is thought that since glutamine is involved in a different number of pathways, inadequate amounts of glutamine can cause stress on cells because of a lack of nutrients. The various pathways that are involved in glucose metabolism are shown in figure 4A. This figure shows many molecules that glutamine plays a role in synthesizing and the pathway by which it is synthesized. One explanation that is offered by the authors is that since glutamine has an effect on nucleotide synthesis, it indirectly has inductive effect on cell death if there is a nucleotide deficiency.
Figures 4B and C show experiments done to determine whether glutamine depletion affects apoptosis indirectly as just stated or possibly directly affects mechanism of apoptosis. In this figure, the authors were looking to see if apoptosis was induced by protein synthesis. In 4B, cells were incubated with OHT present, and then incubated again in a media deficient in fifteen amino acids. The results showed that this depletion of amino acids did not show cell death, suggesting that insufficient amounts of amino acids and consequently inefficient protein synthesis was not the reason for apoptosis in glucose depleted cells.
Figure 4C is a different experiment done to determine the mechanism behind glutamine depleted apoptosis. The author prefaces the experiment by explaining mTOR. mTOR is a kinase that is involved in many cellular processes and is regulated by glutamine. S6 is a target of mTOR. Rapamycin is an mTOR inhibitor. If mTOR activity plays a role in apoptosis with glucose depletion, it would be expected that S6 (mTOR target) would be suppressed by Rapamycin and cell death by glutamine depletion would be inhibited. The chart shows that rapamycin does not however how any effect on inducing apoptosis. All of these factors lead to the theory that mTOR activity does not play a role in apoptosis in this situation since its inhibitor did not prove to affect the cells.
The next experiment shown in figure 5 tests the effects of glutamine depletion on apoptosis when there is a deficiency in the amount of ATP present. The authors were looking to find out whether it was correct that ATP deficiency is the mechanism which was inducing glutamine depleted apoptosis. In order to observe these effects on ATP concentration, the authors used ER-MYC-C9DN cells. Remember from earlier that Caspase-9DN is an intrinsic pathway inhibitor, but does not affect steps leading up to apoptosis. The graphical representation is shown after incubation for twenty four hours and then another eighteen hours in media. After removal of glutamine from the cells, the results showed a minimal decrease in the concentration of ATP (measured in mmoles/ mg protein). There was also a substantial drop in ATP concentration in the presence of 2 deoxyglucose and antimycin a. This drop off is only observed when these two are used in conjunction, but not individually. 2-DG is a glycolysis inhibitor and antimycin a inhibits mitochondrial respiration. When you look at figure 5B, you notice that although antimycin a and 2 deoxyglucose had a substantial effect on the concentration of ATP in the cells, they did not have nearly as much effect on cell death as depletion of glutamine. This is apparent by observing that glutamine depleted cells showed about 80 percent of cells stained with annexin. 2DG and antimycin A cells only showed approximately 30% of cells with annexin staining. Because the addition of 2DG or antimycin a alone showed no drop off in ATP concentration, it is thought that glycolysis or mitochondrial respiration alone can provide the cell with adequate amounts of ATP. After observing these results, the researchers found that apoptosis was not occurring because of inadequate amounts of ATP.
The next hypothesis that was proposed and tested was that glutathione depletion was causing apoptosis. The reasoning behind this hypothesis comes from the knowledge that glutathione’s precursor is glutamine and glutathione concentrations are regulated by MYC. Synthesis of glutathione from glutamine is diagramed in figure 6A. The authors affirmed that large amounts of MYC could provide ample amounts of ROS, which could lead to damaged DNA and eventually apoptosis. Another molecule that plays a role here is l-buthionine sulfoximine (BSO) which is also shown in 6A. BSO inhibits the synthesis of gamma-glutamylcysteine which is also a precursor of glutathione and affected by glutamine. As in previous experiments, glutamine was depleted and the results graphed in 6B. Both lower amounts of glutamine and the addition of BSO diminished the concentration of glutathione. So far, these are the results that were expected as implied by the hypothesis and goal of the experiment. Yet, the results in figure 6C do no support this hypothesis. Activated MYC cells did not show an increase in apoptosis in BSO media. Because BSO is an inhibitor of glutathione but BSO does not induce apoptosis, these results would suggest that the glutathione deficiency by itself does not cause apoptotic cell death.
DNA damage is also a common reason for apoptosis. The next experiment tests whether glutamine depletion damages the DNA. They tested to observe if the reduction of glutamine induces p53 concentrations and H2AX histone phosphorylation. The thought process behind this goes back to figure 4A, which shows that the N-terminal amino of glutamine is involved in nucleotide synthesis. Lack of proper synthesis could possibly lead to damaged DNA which as we know is a common cause of apoptosis. Figure 7 illustrates the results when cells were incubated in media with DON, 6-methylmercapto-purine riboside (MMPR) and etoposide, all of which cause DNA damage in different ways. MMPR is known to inhibit nucleotide synthesis as in the case of the n-terminal amino of glutamine. In 7A, activation of ER-MYC shows increased percentage of cells with chromosomes condensed when there is insufficient glutamine and also in the presence of DON, MMPR and significantly increased with Etoposide. In all three parts of figure 7, you can see that the amount of p53 concentration is increased as the ER-MYC cells are activated. An important observation to be made is that glutamine depletion resulted in a lower concentration of p53 as seen in the charts. The authors also explain that in another paper, etoposide induced phosphorylation of the H2AX but glutamine depletion did not. Using these results in conjunction with the results from this figure, the authors concluded that insufficient amounts of glutamine alone does not result in damaged DNA. This is due to low amounts of glutamine not increasing p53 and not resulting in the phosphorylation of H2AX which would lead to DNA damage and apoptosis.
The Krebs Cycle could also have affects on apoptosis if there is a deficiency in the cycle. The result of the Krebs cycle is a production in NADH. Its intermediates also play pivotal roles in metabolism which is why the authors proposed that the Krebs cycle deficiencies could lead to apoptosis. To test this, the ER-MYC cells were incubated with and without OHT as in previous figures, and then incubated in media with Pyruvate and Oxaloacetate. Figure 8A shows that cells in pyruvate media with no glucose and with OHT showed decreased annexin staining. 8B shows that oxaloacetate showed the same results, which is expected since pyruvate is often a precursor to oxaloacetate. Figure 8C nevertheless, exhibited that pyruvate or oxaloacetate did not increase the number of cells. It was thought that since the amount of NADH, especially in comparison to NAD, would affect apoptosis and NADH is oxidized indirectly by pyruvic acid via LDH, apoptosis may occur. Figure 8D shows otherwise. In this figure the NADH to NAD ratio is shown as a function of time (in hours). The results show that this ration was not affected by glutamine depletion. Therefore, they concluded that pyruvate as a substrate of LDH oxidation of NADH prevents apoptosis but does not increase the number of viable cells.
The decrease in annexin staining caused by pyruvate and oxaloacetate, referred to the in the paper as the “rescue†was still not completely understood. So the next test that the authors performed was to determine whether or not glutamine depletion had negative effects on Krebs cycle production. A system using capillary electrophoresis and mass spectrometry was used to measure metabolite concentrations. Cells were collected at t=6, 12, 18 hours. As in all previous experiments, cells were incubated with and also without OHT present to activate ER-MYC. We now look to figure 9A which shows that activated ER-MYC cells without glutamine all showed lower concentrations of their metabolites. Glutamine metabolites, glutamate, 4-aminobutyrate, 5-oxoproline and aspartate showed this decrease as well as fumarate and malate which are intermediates in the Krebs cycle. The observed findings suggest that glutamine depletion did have inhibitory effects on the Krebs cycle. This leads to the theory that removal of glutamine from the cells induces apoptosis by inhibiting Krebs.
The final experiment wanted to examine why previous experiments done in other laboratories (Shim et al., 1998) showed glucose depletion inducing apoptosis in rat cells, but the findings in this paper did not find that to be true in human cells. Their hypothesis was that the difference in species and cell lines leads to a difference in nutrient requirements. In this experiment, human fibroblasts in foreskin were compared to those in kidney cells. The results are shown in figure 10. Foreskin fibroblasts showed the results that you would expect after learning about the previous nine experiments: ER-MYC cells activated by OHT after removal of glutamine showed an increase in condensed chromatins suggesting apoptosis. This is seen in figure A. Figure B also shows that glucose depletion in the foreskin induces apoptosis, shown by the increase in annexin staining. On the other hand, kidney epithelial cells were only affected by the diminished amounts of glucose and not glutamine. Figure C shows that activated cells showed an increase in condensed chromatins when glutamine is present and no increase when glutamine is not present. These results are also shown in figure D. Figure E shows activated and inactivated ER-MYC cells showed a higher percentage of cells with condensed chromatin when glucose depleted.
