Watch this on-demand webinar presented by Dr Cristina Muñoz-Pinedo
Review the interplay between cell death and cell metabolism, as well as the mechanisms of cell death during cancer and ischemia.
About the Presenter
Cristina Muñoz-Pinedo carried out her graduate studies at CSIC in Granada, Spain, where she studied how glucose regulates apoptosis induced by death receptors. She underwent postdoctoral training in Doug Green's laboratory at the La Jolla Institute for Allergy and Immunology and St. Jude Children's Research Hospital.
Since 2006, Cristina has been led the Cell Death Regulation Group at the Division of Molecular Oncology in the Bellvitge Biomedical Research Institute (IDIBELL) in Barcelona, Spain. The main interest of her team is to understand why and how cells die when deprived of nutrients in the context of cancer and brain ischemia.
- Crosstalk between cell death and cell metabolism
- Targeting metabolism to kill cancer cells
- Cell death induced by metabolic stress
- Ischemic cell death
CM: I would like to thank Abcam and, of course, our attendees this early morning, or late afternoon wherever you are. I would like to talk today about the metabolic regulation of cell death. This is another view of the talk, in which I will talk about the crosstalk between cell death and metabolism in all different aspects. I will talk, for instance, about how metabolism is inactivated during cell death, in particular during the apoptotic process. I will also talk a little bit about how metabolism or nutrients, or the metabolic state of the cell, energetic state of the cell alters cell death pathways. I will also speak about how to target metabolism in order to kill cancer cells. Regarding mechanisms of cell death, I will mention some data as well. The starvation and how this induces different forms of cell death, depending on the cell type; and a few slides about ischemic cell death in the brain.
So what is cell death? As you probably know, apoptosis is the main process, the one that we know more about, and it's caspase-mediated. It means that it's a regulated form of cell death that is mediated by proteases called caspases that dismantle the cell.
On the other hand, so we have CICD which is a caspase-independent cell death, which is still regulated by these same proteins as apoptosis, which are the Bcl-2 family members. But for some reason, the caspases cannot be activated, of course, in post-mitotic tissue. Necrosis that we used to refer to as any form of cell death that we didn't know how it occurs, has actually been shown recently to be mediated by some proteins. With this, we can define several forms of necrosis and, for instance, necroptosis which you had a seminar about, which is a form of cell death mediated by RIP kinases. On the other hand, necrosis can be mediated by the mitochondrial permeability to transition pore, and this is important under some conditions of ischemia. We also have a form of cell death called autophagic cell death, and that is very important for the development of Drosophila and has also been shown to occur in mammals, in mammalian cells; but I'm not going to talk about it today.
What is apoptosis? Apoptosis is a form of cell death that, of course, with clear morphological characteristics that you will probably have seen in cell culture, which are blebbing and cell disintegration. So, biochemically, apoptosis occurs through two main pathways: one is the death receptor pathway, by which a death receptor such as TNF aggregates thanks to the interaction with this ligand. Then this brings together a molecule, an adapted molecule called FADD that activates a caspase. But this caspase, which is caspase 8 in this case, does not heal the cell directly, it acts through activating downstream caspases, that is caspase 3 or 7 that are the executioner caspases. On the other hand, any other stimuli that you can think of, for instance, growth factor withdrawal, or, for instance, in DNA damage or any other form of stress will activate Bcl-2 family proteins to induce the mitochondrial pathway. The mitochondrial pathway, of course, when the BH3-only proteins or the Bcl-2 family activate Bax and Bak that make pores in the outer mitochondrial membrane, and this leads to the release of cytochrome c, which then activates the apoptosome and the initiator caspase 9. Again, it's not caspase 9 that killed the cells, but caspase 3.
So we have two main pathways of which the most important one in terms of the number of cells that are dying every day in our body, or in diseases is the mitochondrial pathway, which is controlled by Bcl-2 family proteins. I would remind you how these proteins work. So Bcl-2, Bcl-xL and CL-1 are anti-apoptotic proteins that are holding, can hold inactive Bax and Bak. This is a simplified model, of course, of how this occurs and there are very complicated interactions between Bcl-2 family members. Bax and Bak are the ones that when active form the pores that leads to cytochrome c release. Now, whenever - BH3-only protein, in this case Bid, but it can be Bim, Puma, Bik, Noxa, lots of the BH3-only proteins are known now. They can inactivate the anti-apoptotic proteins, so then Bax and Bak are free, and also directly activate Bax and Bak to form the pores and leads to cytochrome c release. How is metabolism linked to cell death? We know from a few years ago, by work in the Green's lab that mitochondria are actively inactivated during apoptosis. On the one hand, as you know, mitochondria release cytochrome c and this is the beginning of the mitochondrial pathway. This could lead to an impairment of respiration, but only long-term. But short-term mitochondria can release the cytochrome c and still function for a while.
So once they have caspases activated because of the cytochrome c release, then the caspases go back and they activate, inactivate the mitochondria by cleaving a particular substrate over the respiratory chain, which is called p75. So it's actually caspases that are responsible for the mitochondrial membrane potential drop, and because we know that cells can keep respiring for a while because caspases are inhibited. That it's actually caspases that are responsible for the generation of mitochondrial reactive oxygen species, due to impairment of respiration. This is in more detail what happens: this is the respiratory chain by which cells obtain ATP, and once caspases are activated they cleave substrates. Jean-Ehrland Ricci in the Green lab identified the substrate in complex 1, called p75, and so mitochondrial stopped respiring. But this is not just an epiphenomenon, something that just occurs during cell death using apoptosis, this plays a role in how cells die or more precisely in how fast cells die.
You're going to see here videos of HeLa cells, but in this case the HeLa were expressing the protein p75 that I mentioned from the respiratory chain. In green, you see cytochrome c coupled to GFP, which is inside the mitochondria and that would be released from mitochondria during apoptosis. These cells have been treated with actinomycin D, to induce apoptosis, and we checked several and they were all the same. So after cytochrome c is released you will see that the cells take, bind annexin, because that binds phosphatidylserine, and then they become permeable to propidium iodide. Here you see how cells release cytochrome c, and very early afterwards they bind annexin and very early afterwards, again, they also incorporate propidium iodide. This is faster than - that actually takes a few hours. However, what happens if we are going to express in these cells the p75 protein that cannot be cleaved by caspases, so mitochondria cannot be inactivated? So mitochondria keeps respiring, the cell is trying to die in a proper manner, but it takes much longer. So even though they've released cytochrome c at the same time, they take much longer in binding annexin and the membrane, the cell membrane remains intact for a much longer time. Meaning that in order for the apoptosis to occur correctly, cells need to inactivate mitochondrial metabolism.
Mitochondrial metabolism is not the only metabolic pathway that is altered during apoptosis, and work from Jean-Ehrland Ricci’s lab has shown as well that caspases also inactivate glycolysis by cleaving proteins of the glycolytic pathway. On the other hand, this other type of cell death that I mentioned that is also regulated by proteins of the Bcl-2 family, caspase-independent cell death. Of course, when mitochondria are opened in the same manner by Bax and Bak, when these cells die, I mean, after the mitochondria has released their contents, cytochrome c can still maintain mitochondrial transmembrane potential and cells can keep respiring for a while. But eventually cells die, and in this context caspase-independent cell death occurs from loss of respiration. So inactivation of respiration is common to apoptosis, and to CICD.
On the other hand, we have the converse effect, the opposing effect, which is the regulation of cell death by the metabolic status of the cell, metabolic state. Work from many groups have shown that metabolism or the metabolic status of the cells, or the amount of nutrients in the medium in which cells are grown alters, regulates many cell death pathways. For instance, enforced glycolysis can maintain the levels of the anti-apoptotic proteins and they are Mcl-1, and that is not degraded. While, at the same time, keeping Noxa inactive, this will mean that cells are in a more broad survival state when their glycolysis is enforced. On the other hand, when we don't have enough glucose in the medium, cells can induce the several killer Bcl-2 family proteins, such as Bim, Puma and Noxa, or activate the Bad to either kill the cells or at least maintain them in a more pro-death status. On the other hand, glucose deprivation can also alter how cells die by death ligands. In part, at least, because of the inactivation by degradation of the protein Flip, which is not sensitized enough, so it's not protecting the cells from formation of the death-inducing signaling complex.
Not having enough glucose can sensitize cells to drugs by maintaining the levels of Mcl-1, and regulating some Bcl-2 family proteins, but also to death ligands. These are experiments that we did a long time ago, in which we observed that hematopoietic cells die more when they are treated with death ligands in the presence of the glycolytic inhibitor 2-deoxyglucose. This inhibitor impairs glycolysis and also other things, and when cells are incubated in its presence they are much more sensitive, much more sensitive to cell death induced by death ligands, in cells that are almost resistant to those ligands can be turned sensitive when we block the glycolytic metabolism using 2-deoxyglucose. In fact, this occurs from the very early stages of the aggregation of the death receptors. So when cells have been incubated with 2-deoxyglucose, or without glucose that produces the exact same effect, at a very short time after incubating the cells with a death ligand, in this case its trail, the formation of the complex is dramatically enhanced. The complex that occurs in the membrane that contains Fad and caspase 8, so cells are primed to die from the very beginning, from the very first steps.
As you can see, incubating the cells without glucose can lead to acceleration of every process during the death receptor pathway activation of caspase 8, the activation of caspase 3, the release of cytochrome c and that, of course, in the cells as a consequence of treating with death ligands. Of course, with many death ligands and in many cell types, and have described as well in the laboratory of Jean-Ehrland Ricci as a consequence of cells having less Mcl-1 that can also promote cell death induced by death ligands. On the other hand, these are some observations also from a while ago in which the same cells than before that are hematopoietic cells, the U937 cells. When they are incubated without glucose they are for some mysterious reason, more resistant to treatment with genotoxic drugs such as etoposide or fluorodeoxyuridine, which is a derivative of fluorouracil and it impairs thymidine synthase. So cells are less sensitive to fluorodeoxyuridine, which you can see as well, because there's less cytochrome c release under these conditions.
So having or not having glucose in the medium can alter metabolism in many different manners. The metabolism means very many pathways, but just a pathway of glucose metabolism is sufficient to regulate many molecules of the cell death process. I suppose many of you have logged-in to the seminar, because you're interested in targeting metabolism to kill cancer cells. This is a new area, but it's also at the same time very old, since the observations of Otto Warburg, that we have been thinking of targeting metabolism, because cancer cells have a different metabolism from non-proliferating cells. This is summarized here, again, it's a supersimplification, as you can imagine, there are thousands of metabolic enzymes, but this is a summary of the metabolism of a proliferating cell and a cancer cell. So these cells take a lot of glucose compared to non-proliferating cells; they take a lot of glucose, some of which is derived, is released to the medium after metabolism in the form of lactate and their anaerobic-like qualities. Some of it does enter the mitochondria, tumor cells are not efficient in mitochondrial processes. They still respire and they still can use the mitochondria to produce fatty acids, meaning that many cancer cells make their lipids, their fatty acids from the beginning, from glucose they can make it the novel from to go. Some of them, of course, take them from the medium, but some of them can make them the novel.
Tumor cells can also use a lot of glucose through the pentose phosphate pathway to make nucleotides. So these cells that are proliferating very rapidly, they, of course, need lots of nucleotides. Some of them can be taken from the medium, but some of them are taken, are made the novel from glucose and from some amino acids. Of course, they need a lot of amino acids and glutamine is a particularly important amino acid that is used by many cancer cells that are highly dependent on glutamine. Glutamine can be used for protein synthesis, of course, but it can also be used as the metabolic substrate in the mitochondria. The difference between cancer cells and non-proliferating cells is that cancer cells are using glucose, glutamine and nutrients to make more mass, to make more biomass instead of using them to produce ATP, which is the way that we studied metabolism, which glucose was supposed to be used mainly to make ATP through respiration and Krebs cycle in the mitochondria.
Cancer cells need a lot of nutrients, but to make biomass and this is the main difference. There are selective differences in many cancer cell types between the non-transformed counterparts, and their transformed counterparts. Many other differences and in particular metabolic pathways. This is actually due to the way cancer cells are wired, but just from the beginning, meaning the metabolic changes are intertwined with transformation, they're part of the transformation process itself. Because as part of the starting to grow, they need to rewire the metabolism and here you have the example of oncogene-like protein, like HIF1. HIF1 is usually induced under hypoxia, but many tumors have overexpression of HIF1. So HIF1 can also be activated downstream of oncogenic pathways, such as RAS or PI3 kinase and AKT pathway, or mTOR. The activation of HIF1 rewires the metabolism by regulating virtually every protein of the glycolytic pathway, and including the conversion of pyruvate to lactate. So this is anaerobic glycolysis that cancer cells can do.
On the other hand, HIF1 prevents the use of pyruvate in the mitochondria through respiration, by regulating proteins that regulate pyruvate intake and utilization in the Krebs cycle. So just having HIF1 overexpressed will regulate the metabolism of the cells downstream of many oncogenes. Here is another example of how a protein that is altered, or a complex that is altered in many cancer cells, the mTOR complex regulates metabolism. Again, mTORC1 is activated downstream of many oncogenic pathways, the growth receptor pathways, RAS, PI3 kinase, AKT, all of these will end up hyperactivating mTOR, and this occurs in most cells, in most cancers. So mTOR primarily enhances protein synthesis, but it also leads to activation of many metabolic pathways. It promotes lipid synthesis, it promotes nucleotide synthesis and it also promotes the glycolytic pathway. Again, just the transformation of a cell into a cancer cell by oncogenes, is sufficient to change to rewire their metabolism. Thanks to the metabolic rewiring, cancer cells are much more dependent on nutrients and, for instance, cells that have overexpression of m4c1, because, for instance, they lack this protein TSP. They are much more sensitive to not having glucose in the medium, and this is the property that we want to use in order to target cancer cells by targeting metabolism.
I am not going to talk about every metabolic pathway, but you have this review which is very comprehensive in which they give an idea of how many different metabolic targets we—we, meaning the academics and companies—are thinking about targeting, in order to kill selectively or as selectively as we can, cancer cells. We are thinking of targeting glucose transport by blocking glucose transporters. This suggests a few examples, hexokinase that this is one of the targets of the molecule 2-deoxyglucose that I have mentioned. Also, other transporters that are essential to maintain the pH of the cell, in cells that are producing loads of lactate. Also, lactate excretion, this is very important because cells will die if they accumulate too much lactate and their pH drops. Glutamine uptake and metabolism, there are, again, many, of many people working on how to target the metabolism of glutamine, which is essential for some types of tumors. We should not forget that there are a class of anti-metabolic agents that have been used for decades in the clinic. These drugs such as methotrexate that were known as anti-metabolic drugs, are drugs that block nucleotide synthesis.
These drugs are, of course, drugs that target metabolism, so they are already in the clinic, it's not that we're going to start from the beginning to try and target metabolism, these have been used for decades and some of these drugs are still used in the clinic. Fatty acids are also possibly, probably a good target for many cancer cells that are very dependent on the synthesis, the novel of fatty acids for lipids.
I'm going to talk to you about some work from the lab about starvation, which is starvation can be complete starvation or starvation of a selected nutrient, or even the formal cell death, even the cell death that can occur when we target metabolism. So the idea is to use cancer metabolism to target it, but to kill the cancer cell, not to produce cytostasis, but to be cytotoxic. What we're interested in the lab is understanding how cells die when you target the metabolism, in order to kill them. So, as I mentioned before, if cells have hyperactivation of the mTOR pathway, for instance, they are more sensitive to cell death induced by low nutrients and this is, of course, with many oncogenes.
What happens to a cell that is low on nutrients? For instance, they're incubated with low amino acids, or low glucose. What we will start is a series of reactions by which the cell senses the lack of nutrients and their energetic stress, and they activate this cell, we call a metabolic checkpoint activation of AMPK, inactivation of mTOR, activation of p53. This checkpoint is designed to maintain survival of the cells under conditions of low nutrients. For instance, there's induction of autophagy, fatty acids, oxidation is an alternative way to produce ATP. There's also activation of the unfolded protein response. There's damage to the ER, to the endoplasmic reticulum. In general, all anabolism is reduced, but if something goes wrong and the cell is forced to proliferate, for instance, because it has the oncogenic activation that makes them hyperproliferative, they die. This form of cell death can occur in many different manners, it has been described to be caspase 8-dependent, so this is work from our lab. Hematopoietic cells die usually through the mitochondrial pathway when they are deprived of nutrients, and the nutrient that has been more studied is glucose.
So hematopoietic cells, hematopoietic tumors usually die by activation of BH3-only protein that can be different, depending on the cell type, and then activation of the mitochondrial pathway. We also know that some cells die by necrosis, and this is a supersimplification of the morphology of apoptosis versus necrosis. This necrosis that may be mediated by reactive oxygen species, it's not clear. As I've shown you before, glucose deprivation alters the mitochondrial pathway by regulating many Bcl-2 family proteins. So this is a summary of work from many people and many labs around the world that have shown that deprivation of glucose, or antiglycolitics downregulate Mcl-1 and activate Puma, Bim, Noxa or Bad to kill cancer cells. So when glucose is absent from the medium, these proteins are activated as the cells die through the mitochondrial pathway. There are various signals that can activate these Bcl-2 family proteins, so they die through the mitochondrial pathway.
In fact, we have observed that the treatment with 2-deoxyglucose, which is the most widely we use and the metabolic antiglycolitic drug, is also dependent on the classical mitochondrial pathway. Cells die and by condensing their chromatin, this is the classical hallmark of apoptosis with activation of Bax and Bak. The overexpression of proteins such as Mcl-1 or Bcl-xL, which are anti-apoptotic Bcl-2 family members, protects the cells from cell death. So these cells die by apoptosis, caspase-mediated when treated with 2-deoxyglucose, these are rhabdomyosarcoma; rhabdomyosarcoma cells which is a childhood tumor. We know that it is this apoptosis among many other things, because a caspase inhibitor such as the drug Q-VD prevents cell death. However, we have observed that these same cell lines, the rhabdomyosarcoma, and not only these ones but other ones, although they die by apoptosis when they are treated with 2-deoxyglucose, and here you can see how the overexpression of Bcl-xL prevents cell death when they're treated with 2-deoxyglucose. The same cells when they are deprived of glucose, do not die in the same manner.
We know this because overexpression of Bcl-xL does not prevent cell death, meaning that the mitochondrial pathway is not involved in death induced by glucose deprivation in the same cells that die in a mitochondrial-dependent manner by 2-deoxyglucose. This is two different types of rhabdomyosarcoma and, again, you can see here glucose deprivation kills them in a caspase-independent manner, because we used the caspase inhibitor Q-VD which is something like a Z-VAD, but more specific. It does not prevent cell death, while it prevents cell death induced by 2-deoxyglucose. The same, or more or less the same, although to a different extent in these cell lines which dies mostly in a necrotic manner by glucose deprivation, and in a complete, so pure apoptotic manner when cells are treated with 2-deoxyglucose.
In the same cells, glucose deprivation and 2-deoxyglucose kill the cells in different manners, so we should be careful when using 2-deoxyglucose to mimic glucose starvation, because they're not the same stimuli. Even though they do regulate several Bcl-2 family proteins in the same manner, both with 2-deoxyglucose and glucose deprivation, and Mcl-1 is downregulated, Noxa is induced along time points.
In fact, we've shown that Noxa is responsible, at least in part, of killing the cells after treatment with 2-deoxyglucose. So when we downregulate Noxa, but not Bim, we can see that death is partially prevented. However, this is when we take the cells with 2-deoxyglucose, but, again, the same cells, Bh4 cell line, when they are treated with siRNA against Noxa, and Noxa is downregulated, there's no difference in how cells die in the absence of glucose. So 2-deoxyglucose kills the cells through the mitochondrial pathway, but glucose deprivation kills them by necrosis. This is a different cell type, these are cells now that don't have Bax or Bak so it's deficient MEFs in this case. If MEFs are deficient in Bax and Bak, there's no way that they can do the classical mitochondrial apoptosis, because they cannot open the port that leads to the release of cytochrome c. So we observed that when cells were deficient in Bax and Bak, glucose deprivation would still kill them. They kill them and many tests showed that this was actually apoptosis.
This was surprising, so how can cells die by apoptosis induced by glucose deprivation, if they don't have Bax or Bak? This is supposed to activate the mitochondrial pathway. Well, what we observed that was that caspase 8, what they initiated was caspase in this case, when we used a pool of siRNA against caspase 8, or we make individual clones targeting caspase 8. We can see that cells are protected from apoptosis, they're protected from caspase 3 activation and for many apoptotic events. This is a project that we are still working on trying to understand how caspase 8 is activated in response to glucose deprivation. But what's upstream of cell death? What's upstream of caspase 8 activation or activation of the mitochondrial pathway? We know that glucose deprivation can induce a variety of effects from loss of ATP, interference with lipid formation, interference with many metabolic pathways depend on those phosphate pathway. But we observed that one event is common to cells that are dying by glucose deprivation, or by treatment with 2-deoxyglucose and this is the endoplasmic reticulum stress which leads to the activation of the unfolded protein response.
This is a common event during starvation that there are problems in the endoplasmic reticulum, because, for instance, protein cannot be made at the appropriate rate, because there's not enough amino acids. Or, for instance, if proteins cannot be glycosylated, so these activate a series of centers or in the endoplasmic reticulum that would lead in the end to regulate metabolic genes, and also regulation on chaperones to improve, try to improve and restore the homeostasis in the endoplasmic reticulum. One of these pathways, the PERK, ATF-4 pathway, have been shown to induce cell death. We wondered whether this pathway was also activated by not having glucose, or by treating the cells with 2-deoxyglucose, and whether it was related somehow to cell death. Indeed, what we observed is that both treating the cells with 2-deoxyglucose, treated and treating them with 2-deoxy-… Sorry, glucose separation, 2-deoxyglucose, in both cases the ATF-4 pathway is activated to a different degree. Chop, which is a downstream target of ATF-4 is also induced, and these proteins have been shown to lead to cell death in many systems.
What we have done is prevent the accumulation of ATF-4 either in cells treated without glucose, or in cells treated with 2-deoxyglucose. We have observed that under both conditions ATF-4 is responsible for cell death, meaning that even though glucose deprivation and 2-deoxyglucose kill in different manners. In both cases, cell death is mediated by ATF-4 and so it's really a consequence of the endoplasmic reticulum stress that cells are undergoing, not really because of the loss of ATP that they're surely suffering, or problems with any other pathway. So we have to - I have to remind that this rhabdomyosarcoma and it is possible that other cell types die in different manners. But in our hands in every cell line that we have tested cell death by glucose deprivation kills them by activation of the ER stress pathway, and activation of the ATF-4 pathway. Indeed, in the case of 2-deoxyglucose, this has been observed by other groups as well.
I will spend a few minutes talking about this ischemic cell death in the brain, which is an area in which we are working as well. We want to kill cancer cells by targeting metabolism, we want to kill cells, extra cells by targeting their metabolism. But, on the other hand, there are a series of diseases that are associated to precisely too much cell death due to not having enough nutrients, due to some forms of starvation with or without hypoxia. In general, when you have a problem with blood transport, an occlusion of an artery there will be an ischemic process due to not enough nutrients and oxygen arriving to the cells. This is a major problem worldwide in terms of patients. It could, of course, be myocardial infarction, renal ischemia, these are major problems. We still don't understand ischemic cell death very well, but, of course, there is loads of people trying to understand how cells die when they lack nutrients and oxygen.
What we're trying to understand is how neurons die in the case of stroke. So an occlusion of an artery would lead to an area of ischemia. Some of the cells in the brain that are protected by these ischemia, there is no way to recover them. There's some structural lesion due to the occlusion of the artery, and we cannot recover them. But there's also an area that is called the penumbra, in which there is a mild loss of nutrients and oxygen arriving to the cells. In this case, there's a therapeutic opportunity, because cells can recover if we restore the flux to these cells. So over days the area of penumbra, we hope to find a way of targeting this area so that cells in the penumbra area don't die; and we can reduce this area, and we can recover the damage, but for that we need to understand how cells die. In this case, we have collaborated with the laboratory of Professor Prehn in Ireland, in Dublin, and we have found that when neurons, primary neurons are incubated for 45 minutes, with oxygen glucose deprivation. Which is a bit of a confusing term, and this is the term used by many people working in the area, but it's also complete starvation of the cells in a buffer, which are all cells that are also exposed to hypoxia.
Under these conditions Noxa is induced, Noxa is a BH3-only protein that I have mentioned about is induced at the protein. Also at messenger level very rapidly after the recovery, which is the time that will equal reperfusion. So we treat the cells for 45 minutes in this condition, then we mimic reperfusion that would occur in vivo after the lesion is removed, and this leads to an increase in Noxa. However, in the lab of Dr Prehn, they have used mice that are deficient in Noxa and there's absolutely no differences in the way that cells die. Cells from neurons from knocked out deficient mice die, the same death cells that from the wild-type mice. We were a bit disappointed, but we have observed that there's another Bcl-2 family protein called Bmf that is induced after ischemia. It's induced after ischemia in vitro, but also in in vivo in mice after treating with, after subjecting the mice to experimental ischemia. There is induction of this protein Bmf that has been shown to be induced under conditions that affect protein synthesis.
In this case, there is indeed an effect of not having Bmf present, meaning that the knockouts, the mouse-deficient, mice deficient in Bmf knockout mice are protected from stroke. It's a modest protection, but it's one of the possible proteins that mediates cell death using ischemia. The isolated cells are protected from cell death, but the mice are also protected in terms of several measurements of neurological defects after in vitro, after in vivo ischemia. So with this I'll finish by thanking people from my lab, present and past, that have contributed to the work that I have presented today. People from other labs which I have learnt a lot, and they have helped a lot also in different institutions, people which we collaborate and, of course, our funding agencies. With this I will be back in five, ten minutes to answer your questions, and now I will hand you over to Miriam who will present some interesting products.
MF: Thank you, Cristina, for such an interesting and comprehensive presentation. I just want to remind you that you can keep submitting your questions to Cristina to the Q&A panel located at the right hand side of your screen. So hello again, and I would like to take this opportunity to talk to you about some Abcam products and resources that you might find interesting. We would like to invite you to our Cancer and Metabolism Conference that will be held in September in Cambridge, UK. This conference will cover major aspects of metabolic transformation in cancer, and will highlight potential therapeutic approaches to cancer-specific metabolic pathways. This conference has an outstanding array of speakers such as Karen Vousden, Lewis Cantley or Pere Puigserver, and Cristina herself will be there as well. You can find more information on the event page mentioned at the bottom of the slide: abcam.com/Cancermetabolism2015.
If you have enjoyed this webinar and would like to learn more about apoptosis, you can listen to previous webinars on topics such as caspase death or necroptosis in our site, abcam.com/webinars. These webinars are available for free and can be listened on demand at your own leisure, and you can find a full list of future upcoming webinars and events at our page, abcam.com/events.
We would like to remind you that we have a broad range of kits to detect and quantify apoptosis in a variety of sample and platforms. You can detect apoptosis at pretty much any stage of the apoptosis activation cascade, including caspase activation or decrease in the mitochondrial membrane potential. You can find more information about these products on our page, abcam.com/apoptosiskit. As mentioned in the webinar, tumorigenesis often leads to an altered glycolytic activity itself. As Cristina has explained, production of lactate will contribute to acidification of the cellular environment, which can be used to quantify the cellular glycolytic flux. Our glycolysis assay can measure glycolytic activity using a simple 96-well plate fluorescent reader approach, instead of the most commonly used, but more tiresome potentiometric pH approach.
Oxygen consumption rate is also generally used as a parameter for mitochondrial activity, and measure of cellular respiration. Our cellular, extra-cellular oxygen consumption assay uses a simple 96-well plate fluorescent reader approach to quantify oxygen consumption rates in a variety of samples, such as cell or isolated mitochondria. This assay can be used together with a radiometric mitochondrial dye such as GCA1, to detect mitochondrial respiration and changes in the mitochondrial membrane potential simultaneously. We have also available products for measuring glucose uptake in cells using the glycolytic inhibitor, 2-DG, as Cristina has mentioned during her talk. These are non-radioactive kits that use a simple 96-well plate approach, and can be used with a colorimetric or fluorimetric plate reader. The assay can detect uptake from as low as 50 picomole of the glucose and analogue 2-DG so well.
I just want to remind you that in our cancer resource page, abcam.com/cancer, we have collated all the information we have in our website related to cancer research. You can find links to a specific product, protocol tips, pathways to download and information on the latest cancer events. Thank you very much for your attention. Without further delay, I will pass the microphone back to Cristina who is ready to answer your questions.
CM: Thank you very much, and very interesting. Well, we have many questions, but I hope I can answer most of them. So I'll start. They ask when I showed the data on cell death after glucose deprivation or 2-deoxy treatment, how can I tell there is a difference in the type of cell death, because I have only shown PA staining? Well, of course, I have only shown PA staining, but we have many more data. But I have shown data out of the proteins that regulate cell death, so Bcl-xL, for instance, regulates cell death induced by 2-deoxyglucose, but not by glucose deprivation. Caspases participate in cell death induced by 2-deoxyglucose, but not by glucose deprivation. I've only shown PI, but I have shown that in one case an addition of caspases prevents cell death, and in the other case they don't. So this is what we can tell that there's a difference in the type of cell death. But, of course, we have to look at, and have looked at many more morphological markers, but these markers are usually always consequences of caspase activation. So if in case caspases are not active or do not participate, then we can say it's not apoptosis. Thanks for this question.
Now, here's another question regarding your stress regulation of cell death. Is it something known about ATF-4 regulation of Noxa or the BMF? Well, as far as I know, no, it's not known about BMF, but actually ATF-4 has shown to be transcription factor for Noxa, which makes a lot of sense with our data. However, we have knocked down ATF-4 and looked at the regulation of - at the levels of Noxa in cells treated with 2-deoxyglucose. We couldn't find ATF-4 to be these regulators of the levels of Noxa. It may participate in its activation, but not in its regulation. But, yes, ATF-4 is a known regulator of Noxa and also Bim as well.
Another listener asks about the normal function for BMF. Well, BMF is not one of the best known Bcl-2 family proteins, but there are a number of things that, especially work from Andreas Strasser and Andreas Villunger have shown. For instance, BMF participates in cell death induced under conditions that impair translation, which is something that precisely occurs during ischemia. So BMF participates in cell death by hypoxia, and it also participates in cell death by detachment, a process known as anoikis, which is basically when cells cannot adhere to the substrate, and this could be very important in metastasis. BMF also participates in cell death in that context, but I'm sure we'll soon know more functions for BMF.
Another listener was wondering whether when talking about targeting metabolism in cancer cells, can you discriminate between cancerous and just normal proliferative cells, for instance, stem cells? Actually, although this is not part of the questions, but I would worry in particular about hematopoietic cells like active and proliferative T and B cells. Because their metabolism is wired in a similar manner as well, and this is, indeed, a fair question and I think so far we haven't completely solved this problem. Like if the therapy against cancer metabolism is going to be just like any other type of chemotherapy that will also affect other proliferating cells such as cells that produce the hair. Are we going to have the same type of problem, the same type of problem? Well, we don't know yet. Chemotherapy works because tumor cells are more dependent on the processes that they target, but there are still some pathways that are and this actually relates to the next question, do normal cells make the same metabolism processes compared to the cancer cells? So this applies to both questions.
There are actually some cancer cells that have specific mutations in metabolic enzymes, and I haven't mentioned this topic, but this has been shown extensively. For instance, people that work on isocitrate dehydrogenase, IDH, which is a protein that is selectively mutated in some tumors. So this creates a different metabolism in those cells by mutation, so, yes, there are metabolic differences and also they have shown that in many cancers there are deficiencies, for instance, in the lab of Eyal Gottlieb they have shown that there are deficiencies in the fumarate—oh, I cannot remember exactly the name of the metabolic enzymes now that are in the mitochondria—and that caused deficiencies in respiration, and in the Krebs cycle of the mitochondria. In particular, some renal tumors, but they are specific metabolic pathways altered in specific tumors, not in all of them. We should not say that there is a metabolism of cancer cells versus a metabolism of non-cancerous cells. But there are, indeed, some special pathways that occur only in some cancer cells, and, for sure, proliferating cells have a different metabolism than non-proliferating cells.
This is another question, and another question is how can we target glycolysis in cancer cells? Well, what I have shown is that 2-deoxyglucose is one of these molecules that can target glycolysis in cancer cells. It has actually been used in clinical trials, so 2-deoxyglucose is one of these molecules, but many companies, as far as I know, are developing their own inhibitors to target glycolysis, because tumor cells are much more dependent on glycolysis than normal cells.
Another question is: Is there any known mechanism that explains the difference of apoptotic response to 2-deoxyglucose and glucose starvation? That's what we're trying to find out. There is not a known mechanism so far, but we're certainly working on that trying to understand what is the difference between 2-deoxyglucose and glucose deprivation, if they're both inducers of endoplasmic reticulum stress, how is it possible that one induces apoptosis, and the other induces necrosis?
So we are trying to solve this question, because we cannot be sure at the moment whether 2-deoxyglucose can provide something that cells need, and so apoptotic, of course. Or it's something different like glucose deprivation introduces something in the cells that does not allow apoptosis to occur. It's a subtle difference, but we are still not sure of the answer.
Another question is: Does glutamine starvation activate the same cell death mechanism? Well, this has not been studied so extensively, but in the few reports that there are glutamine starvation does activate apoptosis through the mitochondrial pathway. Indeed, ATF-4 has been shown to participate in cell death in some of these cases, so I think we're going to find a very important role of the ATF-4 pathway, and ER stress pathway in death induced by selective nutrients. By the prevention of selected nutrients, or by drugs targeting different metabolic pathways.
There's another question: Can we tell the cell death form by cell morphology of cells enlarged after radiation, does it tell they're necrosis? Oh, no, it's not that simple, and you can check in other Abcam, very nice Abcam webinars that they are more specifically focused on apoptosis. But, no, it's not as simple. During apoptosis, cells certainly shrink and the nucleus suffers very specific changes, but you would need to use also many other markers to see a difference between apoptosis and necrosis. I refer you to several reviews in the literature, for instance, one that Oscar Tirado has just published, with my collaboration in cancer research, in which we specified, which we mentioned experiments that you should do to distinguish cell death by apoptosis, by necrosis, or by other types, especially if you're a cancer researcher. But it's not as simple, especially radiation and these cells are large, if cells are large, it's possibly, probably not apoptosis, because cells usually shrink.
Thank you very much all for your interest, because there are a lot of questions and it's very interesting to try to solve them. There's a question that asks: If we completely block novel fatty acid synthesis, can we kill the cancer? Well, what I can tell you is that we don't know yet. It's possible that, yes, some fatty acid synthase inhibitors can kill some tumors, but this will probably be very dependent on the type of tumor. So not every cancer is dependent on the novel fatty acid synthesis; but, yes, fatty acid synthesis and the drugs that target other steps of the pathway are probably going to be used in the future to kill cancers, but it will be a specific type of cancer.
Another question, and this would be the last question; the next questions I will answer by email later, and this will be the last one today. Is have you looked at the hexokinase localization within cells after glucose deprivation, and 2-deoxyglucose application, especially at any transport of hexokinase from mitochondria to the cytosol, or to a nucleus?
The answer is, no, we haven't done this, but it's an interesting, certainly an interesting thing to look at because probably this is why the question was asked hexokinase can have different functions, depending on its localization. If it binds the mitochondria it can protect; it has been shown to protect from apoptosis now with a hexokinase translocated to the nucleus, I haven't even thought about this possibility. So thank you very much for that question, and the suggestion. Thank you, Vicky.
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