ASU PSOC Workshop, Wednesday March 21st – Friday March 23rd 2012


Mitochondria are deeply implicated in cancer. They act like little powerhouses within cells that control the energy budget. They are also involved in apoptosis – programmed cell death. When cells become malignant, physical and chemical changes occur in mitochondria, and in the way mitochondria signal the rest of the cell and each other. An early observation of cancer, known as the Warburg effect, is that cancer cells prefer to generate their energy by an alternative chemical pathway known as glycolysis. This pathway is better adapted to low oxygen (hypoxic) conditions, and many solid tumors struggle to receive adequate oxygen. Glycolysis involves profound changes in mitochondria, so understanding the role of mitochondria in the context of the Warburg effect, hypoxia, and apoptosis evasion, could prove to be critical in controlling cancer.
Listen to Audio Interviews and Read Transcripts

Workshop Photos

Tom Seyfried presenting his talk "Cancer as a Metabolic Disease"

Discussion continues in tea break

Workshop Participants

Pauline Interviews Jessica Cunningham

David Stowe,Eric Schon and Sidney Hecht

Nicole Moore of the National Cancer Institute discussing PSOC Objectives and Achivements

Pauline and Paul Davies

Group engagement during Tom Seyfried's presentation

Shirley Taylor, Bob Gatenby, Stuart Lindsay, Karen Anderson and Larry Nagahara

Audio Interviews and Transcripts from the Workshop

Interview with Thomas Seyfried

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Dr Thomas Seyfried believes that cancer is primarily a metabolic disease and so should be tackled as such. Cancer cells have high metabolic needs and so by manipulating the energy balance in the body through diet restriction, Dr Seyfried is convinced that these malignant cells would suffer more than normal cells and even be killed. His book ‘Cancer as a Metabolic Disease’ has just been published by Wiley.

Thomas Seyfried: Tom Seyfried – I’m a professor of biology at Boston College.
Pauline Davies: Now Tom this meeting is about mitochondria and its role in cancer, do you think that this is a neglected area?
Thomas Seyfried: Yeah I think it’s a major neglected area. When we know about the origin of the disease as a mitochondrial metabolic disease then there should be a massive focus of attention on this aspect of the disease.
Pauline Davies: But is it known as a mitochondrial metabolic disease?
Thomas Seyfried: For those who know about energy metabolism it certainly is. Perhaps for those who think it’s a genetic disease this might not be apparent. We know actually what the problem of the tumor cell is, we know what its metabolic deficiencies are, we know how to target those metabolic deficiencies without hurting normal cells. It seems apparent that we should be able to manage this disease with that knowledge and we know we can do this if patients follow the right prescriptions on managing these aspects.
Pauline Davies: Well let’s go right back to the beginning, tell me why is cancer a metabolic disease?
Thomas Seyfried: Well, all cancers suffer from the same kind of problem; they have inefficient respiration. The inefficiency of respiration forces those cells to use an alternative fuel which is fermentation, and it can happen in cytoplasm or even in the mitochondria. It’s the fermentation that compensates as an alternative source of energy for damaged respiration. This leads to genomic instability, local inflammation and the features that we see as the hallmarks of the disease.
Pauline Davies: Because cancer cells are growing very rapidly, they need a great amount of energy to respire, to actually grow, and that’s where the stress comes from?
Thomas Seyfried: Well actually they need a great amount of energy because they’re not effectively metabolizing all of the energy in the molecules they take in. Cancer cells release significant amounts of un-metabolized molecules, so in other words, lactic acid and perhaps succinate and other organic molecules that that would normally be fully oxidized in a normal cell, the cancer cells are wasting this, and this is an indication of an inefficient respiratory system. And it’s the fermentation that drives the proliferation of the tumor cells and also it’s the fermentation that makes the cancer cells drug resistant, so if you target the fermentation they will more susceptible to the drugs and far more easy to manage.
Pauline Davies: Can you, in a very simple way, explain why the fermentation actually drives the cancer. Does fermentation give them more energy to do that?
Thomas Seyfried: Well, you know, we have liver regeneration, the division of normal liver cells to regenerate, they’ll actually grow much faster than a cancer cell and they don’t ferment. So there are other cells in the embryo that will use anaerobic fermentation to develop new tissues and systems like this. The cancer cells are locked into a fermentation profile because they have lost their ability to respire. Cells that can respire will stop their fermentation once the cell becomes more differentiated. The differentiation is also controlled by the energy efficiency of the mitochondria, and if that organelle is damaged in any way, it makes it incapable of using respiratory energy, the cells get locked into a primitive form, the way life was on the planet prior to oxygen. All the organisms were highly fermentative and highly proliferative. The cells were highly proliferative cells in a fermentation reduced environment. Oxygen then brought in stabilization and differentiation and this became the result of having mitochondria in our cells. When those organelles become damaged, these cells revert back to a proliferative condition as they were in ancient times before oxygen came onto the planet. And they will continue to do this now even in the presence of oxygen, because the respiration is deficient and cannot stop this fermentation process. So these cells, as long as they have access to the fuels that drive fermentation, which is glucose and glutamine, they will continue to proliferate and it becomes very difficult to kill them.
Pauline Davies: So you came up with some suggestions for actually controlling cancer. What did you say?
Thomas Seyfried: Our approach to managing cancer will be effective against all forms of the disease, because we view the disease as a singular disease of energy metabolism. So they all suffer from the primary inefficiency of respiration, which is coupled then to excessive fermentation similar to the original theory of Otto Warburg. Now knowing that, can we manage the disease? This becomes not an insurmountable problem. The first step you have to do is you have to treat the whole body, not just the tumor. The body has to be brought into a new metabolic state of metabolic stress where the evolutionary programs for our survival have evolved over millions of years, where we can then tap into alternative fuels due to the genomic flexibility that we have in our systems. Once our body gets into one of these metabolically, or I should say, an energy stress condition, which is actually very healthy, it’s not a painful or harmful situation, the cancer cells now become more stressed than the normal cells because they lack the metabolic flexibility. So the first things we do is put the patients in a state of energy stress by restricting the amount of calories they eat. We bring blood glucose down and ketones up. Blood glucose is the major fuel for the cancer cells and most other cells, especially brain cells. But many normal cells will transition to fat ketones, breakdown product of the fat, which cancer cells have great difficulty utilizing. So putting the patient into a global state of energy stress, puts great pressure on the metabolism of the tumor cell while making the normal cells healthy. The mutations that the tumor cells have, makes them restricted in their ability to adapt to this new energy state. Once the patient is in this new energy state, then drugs can be used to target those pathways that are utilized in the surviving tumor cells, again involving glucose and glutamine, the two major fuels keeping cancer cells alive. Once we hit those fuels, we can manage the disease; patients can live a lot longer.
Pauline Davies: So what does it actually mean for a patient? How much do they have to restrict their diet?
Thomas Seyfried: Well this is an important point and this is one of the reasons it’s a stumbling block. Some patients have to realize they have to stop eating for several days, and get their blood sugar down to 55 to 65 milligrams per deciliter and their ketones up to about 3 to 2 millimolar and then they know they’re in the state. So we have clear biomarkers for patients to get into this particular metabolic state. The problem is a lot of patients are reluctant, they have other thoughts, the issue of cachexia always comes up and they say, “How could you have a patient who’s losing weight stop eating?” And as I said, they’re losing weight because the tumor cells are mobilizing glucose from their tissues of fats and protein. So by lowering the glucose in the patient, you are actually killing tumor cells that are releasing those cachexic factors, so you will lose additional weight at the beginning, but then the body will regain weight and become far more healthy. So it’s a whole systems physiology that has to be used, together with those drugs that target the ability to use glucose and glutamine. And there are a few other things that I know about, but that will get the patient in the right track quickly.
Pauline Davies: So really stop eating completely for three or four days?
Thomas Seyfried: Once the metabolic state is reached, then we introduce those kinds of food that continue to keep blood glucose low and ketones elevated, together with drugs that we begin to administer. This combination of a global metabolic stress together with the drugs will eliminate the majority of the tumor cells in a non-toxic way.
Pauline Davies: So what should people do if they feel that they need to actually pursue this course of events? What sort of food should they be eating after starving themselves for three or four days?
Thomas Seyfried: Well it various from one person to the next; people have to know what their own bodies are capable of doing. They just have to measure their blood glucose and keytone levels which gives them an idea as to, you know, does this food help or not help. You know some people just have to stop eating for a week, it sounds terrible but it works, I know it works, we’ve seen many people benefit from this, and then once the drugs are introduced together with the metabolic state, you can take foods that will elevated ketones further, coconut oil, various kinds of fats, in low doses, not high doses, everything is done in very moderate doses, the body must be allowed to heal itself from within putting more and more metabolic pressure on the tumor cells, every person is different. The biggest obstacle to this is the medical establishment is clueless as to how this works.
Pauline Davies: You’ve done your research in mostly animals, is that right?
Thomas Seyfried: Well we based our studies in animals because we saw how well it worked in humans, so we wanted to make sure that what was so effective in humans, could it work in mice? I was happy to see that what was very effective in humans worked in mice, but interestingly enough it doesn’t work as well in mice as it does in humans. So we should really be doing these studies in humans and not mice.
Pauline Davies: So why aren’t we doing these studies in humans?
Thomas Seyfried: It’s totally different than the way people view the disease; the disease is not viewed as a metabolic disease. If you’re not viewing the disease as the nature of what the disease actually is you’re going to be doing things that are irrelevant to the nature of this. I mean there are some people who are cured by the standard of care and current therapies, but they pay a price for that. They have all kinds of other health issues in those who do survive the treatments. And you know, 60 percent of the people treated with cancer do survive. So you have these many survivors but they pay a price for that survival, they’re debilitated in many ways for the rest of their life if they don’t get a recurrent tumor some other time in the future. We want to eliminate that, we want to eliminate the tumor and keep the body healthy, and that’s what our therapy and understanding will do.
Pauline Davies: Why are we not doing this?
Thomas Seyfried: Because the physicians and oncologists are not trained to do this. If they were trained to do this they would be instituting this. This is not part of the medical practice of the field. Cancer is viewed as a very different kind of disease that needs to be treated with toxic chemicals and radiation. No one is talking about the nutritional metabolic approaches to managing the disease because the physicians themselves are not trained in this. If you’re not being trained to do this, how could you institute this, or even understand the principles and concepts? This is a major stumbling block for the improvement of cancer. We’re not going to make any major advances until the physicians in the field understand that this is a metabolic disease.
Pauline Davies: Because people generally look at cell pathways and gene pathways and you’re saying it’s not the problem with the nuclear DNA it’s a problem with the mitochondria that’s the cause of cancer.
Thomas Seyfried: Yeah. I mean the genomic defects you see in the nucleus are secondary downstream effects, they’re red herrings. People are focusing on things that are not relevant to the nature of the disease. We’re wasting a lot of time and money doing this and it hasn’t led to any improvements, and that’s perfectly understandable if it’s not the core problem of the disease. Once you realize that this is a metabolic disease there will be readily available metabolic solutions that will be effective without hurting the patients.
Pauline Davies: What I don’t understand is why people haven’t looked at cancer as a metabolic disease so much in the past. Why are they focusing on the nuclear problems?
Thomas Seyfried: Well that took place over a many year period, it really kind of exploded with the discovery of DNA in the 1950’s as being the genetic material, and you find broken chromosomes in cancer. It was a natural connection to say, “Oh this is the hottest area in biology; cancer cells have broken DNA; everybody’s looking a gene transcription,” all this kind of stuff. It was only natural course of action to go that route. But Otto Warburg had clearly defined what the nature of the disease was many years ago, and that was kind of considered not important for a variety of reasons, but it was the core issue here. Warburg didn’t discuss the issue of metastasis, which we do, and that’s the key aspect of this; you have to realize what the nature of the metastatic cell is and how its metabolism is disturbed. Once you know that, you’ll manage the disease. This is not an unsolvable problem.
Pauline Davies: So is there any way of preventing cancer in the first place? What would you suggest, that people starve themselves for a couple weeks a year?

Thomas Seyfried: Well I don’t like to call it starving because starving is a pathological condition which is very unhealthy. But if you stop eating for three days, two to three days, and see your blood glucose go down and your ketones go up, you already know you’re enhancing the health and vitality of your mitochondrial system. Blood glucose goes down, ketones go up, the mitochondria biogeness – they get new mitochondria. The inefficient mitochondria undergo autophagy, they’re consumed by the cell for the good of the whole. So the body has an internal control system to purge any cell inefficient in its metabolism. The best way would be to one-week fast once a year, would probably be the singular best way to prevent cancer. This is hard for most people, so maybe three days twice a year, something along this. And as I said you dovetail it in with a religious experience for whatever and it maes everybody feel happy. You can do this with whatever culture or whatever religion; it can be worked in for most people. Let’s put it that way.
Pauline Davies: And the final thing I picked up on, you said to me that inflammation follows something going wrong in the metabolism, but often we hear that inflammation is a cause of cancer, you’re saying it is a result of cancer though. Is that right?
Thomas Seyfried: Yeah it’s a combination of both. I mean you have inflammation, these are wounds that don’t heal; they have the potential to go on and become a cancer if it’s not corrected. At the same time when the tumor is growing in the microenvironment it is itself creating an inflammatory situation. So inflammation is a key component of the disease, so targeting inflammation is one way to reduce the probability of progression and the best way to reduce inflammation is again stop eating. You can control inflammation; it’s a very powerful anti-inflammatory, this is why a lot of the religious leaders would go and fast for these long periods of time. It also has a spiritual component. When your brain starts burning ketones, you actually start viewing life in a different way while at the same time killing any incipient tumor cells.

Pauline Davies: Have you ever tried this yourself?
Thomas Seyfried: Yeah people ask me that and the answer is I’ve tried it. How far can I go? Maybe two days, I’m still working. My students have gone a lot longer than I have. But you know if I had cancer, if I had a glioblastoma, I would stop eating for at least fourteen days and then I would take the drug protocol that we advocate because I know this would be my only chance of survival. And I know exactly what we have to do, how low we have to get the blood glucose down. But if you don’t need to do that, why would you do that? I know what to do if I had to do this. It’s like having the tire changer in your car, if you get a flat tire you know how to use it, but you wouldn’t just go out and change tires just for the sake of changing tires. I know what to do and we’re still working to it, but I think it’s generally a healthy thing to do anyway.
Pauline Davies: Thank you.

Interview with Evangelos Michelakis

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Evangelos Michelakis: I’m Evangelos Michelakis, a professor at the University of Alberta in Edmonton, Canada.
Pauline Davies: What have you thought about this meeting so far?
Evangelos Michelakis: It is interesting to bring such a diverse audience together and I think these things can have some unpredictable effects. There are meeting like that that result in weird changes in the field, for example in a meeting like that fifteen years ago, a provocative idea that nitric oxide is a vasodilator was discussed in the meeting and a few years later the guy who proposed that in such a meeting got the Nobel prize, so you never know when you bring together a diverse audience the unpredictable outcomes.
Pauline Davies: And what is your particular area of expertise?
Evangelos Michelakis: I am a cardiologist and a vascular biologist and I deal with a disease that kills a lot of people, it’s called pulmonary hypertension and it shares a lot of features with cancer in that vascular cells proliferate uncontrollably like cancer cells do. So because of the similarities, I moved into cancer research the past few years as well.
Pauline Davies: So are you still learning about cancer?
Evangelos Michelakis: Of course. It’s a huge field and it’s an intimidating field because decades of funding and efforts have resulted in perhaps hundreds of thousands of papers, so it’s impossible to grasp what is known. The good news is that unfortunately most of what we learn does not seem to mean much because we still haven’t made much progress to cancer so the advantage of a newcomer is that it is much easier to bypass biases and look at things with a new perspective, and this is why this kind of set up here is optimal for newcomers.
Pauline Davies: And you presumably think that mitochondria play an important role in cancer?
Evangelos Michelakis: I believe that they play an important role in many diseases including cancer. What I was hoping it will become apparent for meetings like this is whether there is evidence that they cause cancer as opposed to they facilitate cancer growth. What I think most people will now agree is that they play some kind of role. Because up to ten years ago most people thought mitochondria just damaged, just a result of the cancer process and therefore if you look at them as damaged, they’re not therapeutic targets whereas if you start looking at them as playing a potentially causal role, then all of the sudden they become therapeutic targets. And since the field is in desperate need for new therapeutic targets, mitochondria provide an extraordinary opportunity for new therapeutic options.
Pauline Davies: So what do you think is the main way forward in this field?
Evangelos Michelakis: I think it’s to break down all the existing biases and dogmas and start looking at what the data shows us, what the information about the mitochondria shows us and take a multidisciplinary approach. Everything in mitochondria is redox physiology, the way they communicate with the rest of the cell, and most of their impact on the cell has to do with biochemical reactions that can be deduced to a donation of an electron or two, in other words very hardcore physical sciences’ principals. Therefore the contribution of physical sciences and the way to look at the role of mitochondria and similar processes in cancer, or deduce them down into the simplicity of a give or take an electron reaction could potentially offer a new window and perhaps simplify the chaotic status of their knowledge at this point.
Pauline Davies: In fact you proposed three things that you thought needed to be done. I think one of them was to actually ascertain whether or not mitochondria could cause cancer; I know you alluded to that.
Evangelos Michelakis: Yeah I think the one question is ‘Do mitochondria cause or facilitate cancer or are they just innocent bystanders?’ Then number two is, whatever the mitochondria changes in cancer are, are they irreversible, because if you can prove that they are reversible it means they could be therapeutically targeted, and if they’re causally related, then you could have a new therapy for cancer. And the third is that new approaches need to be followed because I’m not sure the traditional biochemistry has given us this information. Keep in mind that there is this new theory for cancer but it hasn’t been based on dramatically new information, it’s just information that was always there, but people looked at it from a different perspective. So now people are just starting to look at it differently and now you have a metabolic theory for cancer being born. So alternate approaches and new ways of looking at the same data is really needed.
Pauline Davies: What did you think about the proposal to try and kill mitochondria by increasing the calcium uptake of the mitochondria and prevent the calcium from escaping?
Evangelos Michelakis: Well that’s one of the many things that mitochondria do, and I’m not sure the best way to talks about that is killing mitochondria because all we care about is killing the cell, the cancer cell or not. So killing the mitochondria doesn’t necessarily mean that you can kill the cell. Perhaps the best approach is to make cancer mitochondria kill the cancer cells, because that’s what they’re trained to do; the mitochondria makes the cell suicide, the phenomena called apoptosis. So how can you make cancer mitochondria cause the cancer cell to commit suicide, but not normal mitochondria? That’s the key question in the field. Because you can kill cancer cells no problem. Number one you kill normal cells too, and number two the cancer comes back because you didn’t do a complete job. So how can you make the mitochondria kill cancer cells? A more radical approach would have been if there’s evidence that mitochondria causes cancer and the mitochondria dysfunction is reversible. If you normalize mitochondria, could you normalize the cancer cells to come back normal cells? And that’s a more provocative and radical approach that I’m not sure we could exclude as a possibility.
Pauline Davies: And finally do you see yourself having any new collaborations as a result of this meeting?
Evangelos Michelakis: I already had potentials for collaborations with Dr. Schon and Dr. Koopman. It was obvious that there is overlap of interests and excitement, so there are at least two collaborations for me that came out of that so far.
Pauline Davies: Thank you very much

Interview with Erik Schon

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Erik Schon: My name is Erik Schon, I am from Colombia University and I was trained as a molecular biologist but I have spent the last twenty-six or twenty-seven years working on human mitochondrial genetics and human mitochondrial disease.
Pauline Davies: And you’ve just called yourself a contrarian, why so?
Erik Schon: Well I am a contrarian on some aspects of mitochondrial DNA mutations as the impinge on cancer as a process and there’s been a rather large body of data accumulating in the last, almost fifteen years, stating that there are mutations in mitochondrial DNA and of course mitochondrial DNA is the information storage of mitochondria and the information storage to make energy in the cell. In any event mutations in those genes have an impact on either the promotion of or the progression of cancer and I have my doubts about that. So my guess is that even though there are a lot of researchers working on cancer, and there’s a minority that work on mitochondria in cancer and a minority of a minority who work on mitochondrial DNA in cancer. In any event, the people who have identified these DNA mutations, at least in my view, have done what I would consider a partial job in explaining what they have found, meaning you can have mutations in mitochondrial mutation in DNA but then it is incumbent upon you to explain what the functional consequences of that are. The literature is actually quite sparse on that topic. But that even begs the question as to whether the mutations in mitochondrial DNA are present in the first place, and without going into the technical details I believe that a lot of the DNA mutations that have been found actually may not be arising in authentic mitochondrial DNA as we understand it.
Pauline Davies: So are you saying the mitochondria don’t have a role in cancer?
Erik Schon: No, mitochondria do play a role in cancer, that’s been the subject for the last two days, and they clearly play a role on metabolic grounds, from the point of view of apoptosis, programmed cell death, from the point of view of the distribution of mitochondria in tumor cells versus normal cells. But what I am saying is that the claim that there are mutations in the genome of mitochondria that affect the ability of mitochondria to make ATP I think are a little bit over blown. Mitochondria might be unable to make ATP but not for the reason ascribed to it. That’s my contrarianism.
Pauline Davies: So what else could be going on?
Erik Schon: Well there is no evidence that genes are being silenced in mitochondria, the evidence that there is epigenetic changes in mitochondrial DNA are all also rather minimal. Right now there’s only one paper on the topic. Fortunately the author of that paper is with us and we’ve been discussing that, but it would be nice to see a replication of that work — that’s what a scientist typically says. So with respect to mitochondrial DNA my gut feeling is that the mitochondrial genome in tumor cells in most cases is fundamentally intact, and that the down regulation of mitochondrial function is arising elsewhere, which is the basis of the Warburg hypothesis, saying that oxidative phosphorylation is down regulated even in the presence of sufficient amount of oxygen which would allow such phosphorylation to take place, and I do not know how it arises. People far more au courant with this are much better able to speculate on it than I am.
Pauline Davies: And what about the suggestion that we heard several times in fact that if we could only normalize mitochondria in cancer cells, then cancer could be stopped in its tracks.
Erik Schon: Actually I’m kind of taken with that idea. So if we buy the idea that mitochondria down-regulate, and I think that is true, perhaps renormalizing it would help. I don’t think it would cure the problem. What might be more useful is what some people at the meeting have intimated is that we could take advantage of the down-regulation as a marker of a tumor cell to distinguish it from a normal cell and then use that metabolic marker as a way to target those bad cells for destruction, or at least for amelioration in the case of metastasis.
Pauline Davies: And what about Tom’s work.
Erik Schon: Tom Seyfried
Pauline Davies: He was suggesting that you up-regulated the mitochondria and then treated a patient with drugs. Do you think that that would be a way forward?
Erik Schon: Yeah Tom’s idea was really quite provocative; I was rather taken by it. So just to rephrase it, I think his idea was to have patients go under caloric restriction before they even had resection of their tumors. The caloric restriction would, we already know this, caloric restriction impinges on mitochondrial function in terms of normal longevity. His idea was to do the caloric restriction and then force cells to use their mitochondria by essentially flooding them with ketogenic precursors. It would do two things; select for normal mitochondria function but perhaps in the context of caloric restriction starve the tumor. It’s a little bit baroque, the biochemistry, and then the tumors would disappear. He showed some data that actually implied that that actually may be working. So why not try that? I’m all in favor.
Pauline Davies: And what have you thought about this workshop overall?
Erik Schon: It’s been terrific. I really like the workshop for the simple reason that you have only a few people, perhaps a dozen or fewer, there’s no compulsion to show off in front of your colleagues because I barely know most of these people, you’ve been given the carte blanche to speak your mind without any kind of retribution coming back. To me it’s what science is all about; the problem is on the table, you attack it in an intellectually honest way, let the chips fall where they may and it’s remarkable how many few times you get that in what we would call real conferences. This is the real conference.
Pauline Davies: Can you see any new collaborations coming out of this?
Erik Schon: I already set one up with Michelakis Evangelos, I was a postdoc in a Greek lab so I have to say Greek names a little better than I do. He’s done some really nice work on hypoxia that intersects with a gene that we have discovered which may actually affect the phenomenon that he’s insecting, so we’re going to collaborate on it.
Pauline Davies: Terrific and good luck!
Erik Schon: Thank you.

Interview with Jack Tuszynski

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Jack Tuszynski: My name is Jack Tuszynski and I’m a professor of biophysics at the University of Alberta in Edmonton, Canada. I’m a computational scientist working in the field of cancer research.
Pauline Davies: What have you made of this meeting so far?
Jack Tuszynski: Oh it’s been phenomenal. I think we’ve had some tremendous talks about the role of mitochondria in cancer and also other diseases such as Alzheimer’s, but primarily the talks were centered around the switch from oxidative phosphorylation, which is a normal mode of energy generation in most cells, to glycolysis which is like fermentation in cancer cells. And there are a lot of facts that play a role in it and some of it has to do with instability or general deregulation of cancer cells, but according to the scientists that were speaking here, the root cause of the problem is actually to be found in mitochondria not in nuclear DNA.
Pauline Davies: Which is a completely different way of thinking about cancer, isn’t it?
Jack Tuszynski: Well exactly and so far I think we have really, nothing in the clinic at least that addresses the problem of returning the cancer cells to normal, metabolic function. And my opinion it is also a game changer, and paradigm shift where instead of trying to eradicate cancer, the cells, or kill them to just put it bluntly, we would try to simply restore them to a normal behavior.
Pauline Davies: And some people suggested way of doing that. There’s one person that suggested depriving the mitochondria of glucose and then treating with drugs. And then we heard someone else suggesting a different kind of drug. How do you put all that together?
Jack Tuszynski: I don’t actually think that this is in contradiction. The first person, Tom Seyfried from Boston College, suggested that before we actually start treatment, cancer patients with pharmalogical agents or radiation or surgery, we should introduce colaric restriction. So maybe a seven-day long starvation diet more or less so that the cells get energized to some degree and a shed some unnecessary balkst and then they will be easier to treat with normal pharmalogical agents. The second talk was actually directed at avoiding chemotherapy all together and instead using a molecule called DCA, which is over the counter used in other disease I think as childhood diabetes, juvenile diabetes, that triggers and effect that naturally restores mitochondria to normal activity. This molecule, what is does, and it has been demonstrated so far in animals and some small studies in phase one clinical trials in humans, this molecule lowers the transmembering potential in mitochondria which then allows to release pro-apoptotic proteins. That means cancer cells in the presence of this molecule can kill themselves, to put it bluntly again.
Pauline Davies: Yeah and in fact I think it was used in treating a buildup of lactic acid, was it? Or preventing it?
Jack Tuszynski: That’s correct. Build up of lactic acid is a bi-product of glycolysis or fermentation and that is a contributing factor to the proliferation of cancer because it creates and acidic, hostile environment in which normal cells don’t thrive very well and cancer cells continue in their incorrect metabolic pathway.
Pauline Davies: All this work seems to me to be very relevant to the potential treatment of cancer. Do you think more oncologists should get to know this field?
Jack Tuszynski: Absolutely. We talked about this and I think it’s probably going to be on knowing the field because what we need to do is address this issue from the FDA approval point of view, from the medical professional point of view where the current paradigm seems to be running out of steam and after forty years of the so called war on cancer we have only managed to improve survival by seven percent. So clearly it’s highly incremental and not very successful so we need to seek different methods.
Pauline Davies: And are you yourself convinced that cancer is a metabolic disease?
Jack Tuszynski: I am and probably all of us here and most of the practitioners know that there are metabolic aspects to cancer, but probably outside of this narrow group, most people think that this is a secondary effect whereas the speakers at this conference believe that metabolism is the root cause of cancer, incorrect metabolism causes cancer and leads to genomic instability and other downstream effects.
Pauline Davies: Will your thinking be changed as a result of this meeting or are you very familiar with this sort of work?
Jack Tuszynski: I’m reasonably familiar with some of it, but I’ve learned a lot of new things. I made a lot of notes and I’m actually going to start a project with my PhD student who’s starting in this area of the Warburg effect, so that’s another historical note that the metabolic cause of cancer was first hypothesized by Otto Heinrich Warburg in 1926 or 1927, almost a century ago.
Pauline Davies: And he suggested what?
Jack Tuszynski: Well he suggested that that is actually the cause of cancer, incorrect metabolism, glycolic metabolism, which increases with malignancy is the root cause and what we should target. And most of the people at this workshop concur and are now actually providing concrete ways of doing it.
Pauline Davies: And cancer biologists now think of the Warburg effect as cells uses anaerobic respiration, is that correct?
Jack Tuszynski: That is correct. And that of course is related to some other issues which we’ve discussed at other workshops here in Tempe, namely hypoxia, so low oxygen delivery to cancer cells as well as stiffness and mechanical defects and radiation defects. So all of this is somehow connected but here we believe that it starts with mitochondria. In my group what we would like to do is put it all together into a quantifiable, mathematical model that shows how this pathway to abnormal metabolic activity can be properly described by equations and therefore modeled and therefore predicted and eventually, in a scientific way, stopped. So without wasting too much time on adhoc procedures and methods we would like to have a very exact way of solving this problem. And I’m sure with Professor Davies we will be able to make progress in this direction.
Pauline Davies: And as a result of this meeting have you formed any new collaborations?
Jack Tuszynski: Well indeed yes. And I was pleased to hear Dr. Koopman’s talk from the University of Nimegen in the Netherlands, his group is working on mitochondria and has been able to develop very sophisticated software that simulates the biochemical reactions taking place within the cell including both glycolysis and oxidative phosphorylation and he very graciously agreed to collaborate and allow us to use his software for applications in cancer and his groups has actually been working on other diseases, not cancer, so it’s a perfect match.
Pauline Davies: Terrific we look forward to those results.

Interview with Shirley Taylor

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Shirley Taylor: My name is Shirley Taylor and I’m an associate professor at Virginia Commonwealth University in Richmond, Virginia in the Massey Cancer Center, and my studies involve largely the control of gene expression through epigenetic mechanisms.
Pauline Davies: Have you been enjoying the meeting?
Shirley Taylor: This is probably the most fun I’ve ever had at a meeting before. I’ve never been to a meeting with only sixteen people but the discussions have been phenomenal.
Pauline Davies: Have you learned a lot?
Shirley Taylor: More than you can imagine. Cancer is not a new field to me, but we focused up till now on the nucleus and what’s happening in the nuclear genome with mutations and silencing of genes, so going into the mitochondria, this is new for me, but is so relevant to cancer. And so I’ve learned so much that I will take home now to my own research.
Pauline Davies: I was amazed to find that there could be thousands of mitochondria in a cell.
Shirley Taylor: And each mitochondrion can contain multiple genomes. Each genome could be doing something completely different, although they seem to contain the same genomic information, that’s what’s also mind-blowing.
Pauline Davies: Think life is immensely complicated.
Shirley Taylor: It really is, and I think we have levels of complication that we’re only just starting to scratch the surface on.
Pauline Davies: So tell me why mitochondria are so relevant to the story of cancer.
Shirley Taylor: The cancer cell has fundamentally changed in its metabolic activities and requirements so it has tended to down-regulate mitochondria and subvert cellular mechanisms to obtain energy. And this down-regulation of mitochondria that we don’t understand, although we understand some of the mechanisms, whereby its up-regulated cytosolic metabolism.
Pauline Davies: What did you say?
Shirley Taylor: Cytosolic, the energy metabolism through glycolysis that happens outside of mitochondria.
Pauline Davies: So what could be going on in the mitochondria?
Shirley Taylor: One of the things that we’ve discovered is that there is a mechanism for changing patterns of gene expression that involves epigenetic modification, that’s changes in covalent modifications on the DNA that probably change how that DNA interacts with controlling proteins. So if you change the patterns of expression of genes that are encoded in the mitochondria, you can actually fundamentally change the way mitochondria does energy production.
Pauline Davies: So somehow the messages from the nucleus of a cancer cell are going into the mitochondria and telling the mitochondria to turn itself off in places.
Shirley Taylor: Maybe not necessarily turn itself off, but change the way it deals with energy metabolism and its own genome, but we also think this feeds back to the nucleus to fundamentally change the patterns of gene expression in the nucleus, and the whole system feeds on itself. Nuclear-mitochondria communication I think is where we’re going to find the important connections.
Pauline Davies: Now if the mitochondria weren’t altered in cancer cells, do you think cancer cells could be so successful?
Shirley Taylor: No, I don’t think they could be as successful as they are. They’re evolving in such a way that they can make the energy that they need no matter what stresses are put on by the immediate environment. They’re going into survival mode by changing their metabolism the way they do.
Pauline Davies: So if we could change the mitochondria back to normal functioning, could that actually help control cancer?
Shirley Taylor: I really do believe that is one approach that’s got to be incorporated into how we treat the cancer over all, but it’s not the only one. One of the things I’ve heard here at this meeting is predominantly on how to reprogram mitochondria to be more normal. If you put that in the context of also treating the genetic part of cancer and the epigenetic part of cancer then I think you almost have a three-fisted approach that will really change how we can manage cancer.
Pauline Davies: So overall the message is the mitochondria have been a neglected field till now and could be very important to help us manage and control cancer.
Shirley Taylor: I really do believe that’s true because I think what it’ll do is it’ll fundamentally change how a cell responds to the nasty things we normally throw at them. If you change that metabolism, you prime the cells, now to be much more sensitive, perhaps to lower and less toxic concentrations of these nasty drugs. So I think if you start at the mitochondria you can change everything that you do in treating cancer.
Pauline Davies: Why do you think this has been a neglected field for so long?
Shirley Taylor: Mitochondria are very difficult to work with. We don’t know as much as we should about how the processes within mitochondria are regulated just because they’re difficult to work with. But that’s changing, and it’s clearly changing from what I see and learned at this meeting.
Pauline Davies: Is that because they’re so small?
Shirley Taylor: They’re small, they’re interconnected, they move, they’re very plastic, they’re very fluid in the way they are distributed through cells and how that changes over time. It’s like trying to pin a snowflake down, it’s changing constantly as the environment changes it. So to try to isolate them, they lose many of their properties because now they’re out of their normal environment, but to work with them within cells is hard because they’re continually changing.
Pauline Davies: So are we now more successful at looking at mitochondria because you’ve got more sensitive tools?
Shirley Taylor: I believe we’re developing more sensitive tools, some of the tools my lab is developing I hope will lead us in that direction, but we’re going to capitalize some of the tools that were presented at this meeting I think in future collaborations. It will allow us to bring those tools to bear on the particular questions that I’m trying to ask.
Pauline Davies: Has it encouraged you to do more interdisciplinary research?
Shirley Taylor: Yes definitely. And I think that the collaborations that will come out of this meeting are really going to be super.
Pauline Davies: And anything in particular? Any collaborations you’re envisioning?
Shirley Taylor: Dr. Werner Koopman from the Netherlands has some probes of energy fluxes through the cell that I think will be really important for us to understand what the downstream functional significance is of our initial findings.
Pauline Davies: Well good luck with that.
Shirley Taylor: Thank you.

Interview with Werner Koopman

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Werner Koopman: My name is Werner Koopman and I’m from the Radboud University in Nijmegen in the Netherlands, in Europe.
Pauline Davies: And what do you specialize in?
Werner Koopman: My specialty is trying to understand how mitochondrial morphology and dynamics is linked to mitochondrial function.
Pauline Davies: And are you thinking about any diseases in particular, or are you thinking about cancer?
Werner Koopman: We mainly work on rare diseases, rare, metabolic diseases. We currently work on isolated complex 1 deficiency, so that’s a malfunctioning of the first complex of the respiratory chain of the mitochondria, but we are pretty confident that the knowledge that we gain in that field will be of broad implications for many, let’s say, more important diseases.
Pauline Davies: So what have you though of this meeting so far?
Werner Koopman: I found it, for the first day now, inspiring also to hear from people from other fields, let’s say, the cancer field in particular. I met already a few people for some promising future collaborations.
Pauline Davies: In fact Jack Tuszynski in Canada, I believe, is hoping to collaborate with you in the future.
Werner Koopman: Yeah we would like to combine our expertise on modeling of metabolism, which we use to understand rare mitochondrial diseases and probably they also have some use for applications for understanding cancer metabolism.
Pauline Davies: Had you ever considered applying your work to cancer before this?
Werner Koopman: I had considered this, but within the constellation of most research projects you are not able to switch to a totally different topic that quickly. But sure, I think the techniques and the strategies that we use for mitochondrial diseases might be of benefit for cancer-related diseases as well.
Pauline Davies: So what do you think of this concept of the PS-OC, bringing physicists and non-physicists together to look at cancer?
Werner Koopman: I think it’s of crucial importance that different disciplines learn from each other’s expertise and not only at the level of concepts, since I think physical concepts are a good way to approach biological problems, but even more I think the tools that physicists have developed can be very nicely complimented by biological tools and in that way you can move forward faster.
Pauline Davies: Thank you very much.

Sidney Hecht summarizes the meeting

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Sidney Hecht: My lab works on projects involving cancer and on one involving mitochondria, but we don’t work on the relationship between mitochondria and cancer. Our cancer program is focused mostly on agents that interact with nucleic acids and nucleic acid-protein complexes. The mitochondrial program is focused on inherited mitochondrial disorders and on making molecules that compensate for dysfunction of the respiratory chain by managing the flow of electrons that have leaked from the respiratory chain. Part of our existing cancer program has led us down a pathway in which we study clinical agents that function by inducing redox stress; that is related mechanistically to what we study in inherited mitochondrial diseases. None of the data I will show you outside the workshop summary has anything to do with what is going on in my lab, but it was influenced by the experiments in my lab. I’m going to show one PowerPoint slide for each of the talks and focus on the clinical implications. The first talk that Eric Schon gave was intended to get us up to speed on mitochondria and things we didn’t know before. One of the things that seemed especially interesting to me is that one of the functions of the proteins imported by the mitochondria includes signal transduction which turns out to be interesting at the level of redox stress. Another interesting point is the notion that mitochondrial DNA fragments can be inserted, or are inserted, into nuclear genes. The implication is that if I’m a mitochondrion and I can get my DNA fragments inserted into nuclear DNA, then the nuclear DNA of the cell in which I reside is potentially under my control. The implications of this conjecture might be worth thinking about.
Mitochondrial dynamics: I found two very useful concepts here. I had not really thought about mitochondrial disorders as being primary and secondary in nature, but it’s entirely logical to consider them that way. Primary disorders can be caused by any of 240 mutations – in 240 different genes. That includes genes involved in reactive oxygen and nitrogen species, and presumably changes in mitochondrial membrane potential. Secondary disorders include changes in ATP levels, the redox state of the cell, the mitochondrial permeability transition, and again effects on signaling pathways. Additionally, we learned of the dynamics of the system, where mitochondrial networks can control metabolic activity through changes in the connectivity of the mitochondria. What I took away from the next presentation was the linkage between mitochondria, oxygen sensing and cancer, and this is very closely related to some of the things I want to share with you after I summarize the individual talks: the mitochondrion is clearly an oxygen sensor. One of the molecules that must participate in that way is HIF-1 which, interestingly, can alter cellular behavior without being under conditions of frank hypoxia. We talk about molecules that respond to hypoxia, but these can be relevant to simple oxygen deficiency, both thermodynamically and kinetically. We also learned that cancer cells have an increased membrane potential, and that this can be reversed by molecules like dichloroacetic acid (DCA). In fact, dichloroacetic acid has been used for treating patients with mitochondrial disorders and appears promising. One of the potential downsides is peripheral neuropathy, which in some circumstances can be extremely serious, but in others perhaps reversible. I’d like to ease into the business of clinical implications. I’ll put on my hat from when I was in the pharmaceutical industry and talk a little about dichloroacetic acid, something I know only from a distance. I’ve read about it in the popular press and heard about it here. I understand that it has some potential liabilities but, at least for some cancers, would also seem to have some potential advantages. In the discussion yesterday we seemed to focus extensively on the fact that DCA is unusual relative to most molecules that advance to the clinical interface. Most of those molecules are owned (through patents) by some organization which has invested into bringing them into the world and wishes to get its investment back while addressing an unmet medical need. In comparison, molecules like dicholoroacetic acid are chemicals that have been in the public domain for a long time. In principle, no one can bring them into the market place and have an exclusive marketing position in spite of having invested a significant amount of money in preclinical and clinical development. Unsurprisingly, this tends to discourage investment, even for very promising compounds. In my view, this is one of the places where government has an important role to play, e.g. by supporting clinical trials involving such compounds. Government can also be involved in other ways. For example, it is possible to create exclusivity through legislation, as has been done for orphan diseases. In this case, the FDA can grant exclusivity to the organizations who invest in creating a novel type of therapy in a given area. In principle, there is no reason that the same thing couldn’t be done for a potential drug like DCA. So the issue of lack of patent protection is potentially manageable, but the solution is not in the realm of science. That’s what I took away from this talk at the level of a potentially new type of drug.
(Much discussion followed about the difficulties of getting people to invest in clinical trials for this drug – it was suggested by a participant that the work could be done overseas in a well-regulated country where the costs involved are lower).
Moving on to the silencing of mitochondrial genes, we heard a very interesting talk about methylation patterns in mitochondrial DNA. Again, I focus on the linkage to p53 which is something I’m going to get back to as we get into clinical implications. We learned that loss of p53 results in selective upregulation of the mitochondrial isoform of DNMT1. In the presentation dealing with bioenergetics we heard a very interesting, very thorough analysis of all of the potential targets for killing mitochondria, most of which would seem not to have been explored in any systematic way to date.
So I’d like to switch gears now and talk about something that my laboratory spends a lot of time thinking about, namely oxidative and reductive stress in mitochondria and their potential effects in causing the progression of inherited mitochondrial disorders. I was curious to know whether or not similar effects had been looked at in studies of cancer. There is a fairly recent review article by Barry Halliwell (Biochem. J., 2007, 401, 1-11) who has studied reactive species for quite some time; it’s an update and extension of earlier reviews he had written. The review, and articles cited therein, does consider the possible linkage between oxidative stress and cancer. There are a number of animal models that have been employed, and more than one reported study in which in one or multiple antioxidant systems have been knocked out in the animal model. The animals frequently exhibited enhanced age-related cancer development, pretty convincing evidence that suppressing oxidative stress is a good thing if you’d like to avoid cancer. Malignant tumors often have increased levels DNA base oxidation which comes from reactive oxygen species. This observation supports the idea that there is increased oxidative stress in tumors; again, this has been observed in vivo. The tumor suppressor protein p53, interesting enough, actually has 10 cysteine residues and one of these cysteine residues has been observed to be modified by nitration in neurogliomas. So very interestingly, we have a potential linkage between an oxidative event (involving a reactive nitrogen species rather than a reactive oxygen species) involving a key protein, and a human glioma. To date most of the correlations of this type have been reported at the level of animal models. At the level of cell culture, many more experiments have been done. Let’s look first at p53 and then at some of those in vitro experiments. It turns out that p53 has both pro- and antioxidant functions. Under normal circumstances, where cells are not excessively stressed, p53 induces antioxidant genes which prevent or repair mutations, facilitating normal cell growth. In cells that have been under excessive pressure p53 is a mediator of cell death, and it does that by inducing prooxidant genes to shut the cell down. The circumstance encountered less often involves mutant p53s , such as the nitrated p53 described previously where we have a different sort of mediator, potentially doing little or nothing in terms of inducing prooxidant or antioxidant genes, but which may allow mutations in cellular DNA to accumulate, thereby facilitating tumorigenesis. This provides an example of alteration of normal cell functions potentially resulting from oxidative stress.
Here is some interesting data from human tumor cell lines. Multiple papers made the point that constitutive levels of H2O2, but not superoxide, have been observed for a number of tumor cell lines. Further, maintaining the transformed phenotype actually required a sustained increase in peroxide levels. That finding was fascinating for me, since I started out to look for mitochondrial linkages to cancer.

Question by Stuart Lindsay: To sustain the phonotype, the concentration of peroxide has to continue to increase?
Sidney Hecht: No, it has to be sustained at high levels – but it doesn’t have to increase continuously.

However, given my interest in possible mitochondrial involvement in cancer, what discouraged me was that at least one study looked at the effect of inhibitors of mitochondrial respiration in cells having high peroxide levels. They employed just about every type of molecule that has ever been used to inhibit a mitochondrial respiratory chain complex, but none of them affected peroxide production in the cells that were being studied. Thus the source of the elevated peroxide in these experiments was not the mitochondria. Now I suppose this doesn’t exclude the possibility that if you had sufficiently elevated H2O2 produced in the mitochondria you could still get the same effect, especially since it is well established that mitochondrially derived peroxide is exported to the cell cytoplasm. But at least in the specific studies cited in the Halliwell reference, there was a source of H2O2 other than mitochondria that was associated with the transformed phenotype noted for the cancer cells. The peroxide levels in these cells, and you’ll see it in another paper that I’ll cite, are really high. They are 10-fold, sometimes 100-fold, higher than in normal cells.

Question by Paul Davies: Does that mean the cancer cells like it or are trying to get rid of it?
Sidney Hecht: It’s not clear. One thing you might think: if they had elevated levels of peroxide, they may be close to the edge. Can you push them over the edge? I’ll discuss a paper in which experiments were carried out to test this strategy. But I hasten to add, as is the case in many cells that are aberrant metabolically, the cells have learned to accommodate to their situation. There is a report in which someone took cells with elevated levels of peroxide and studied what it took to physically lyse the cells with added peroxide; they were very resistant to additional peroxide.

Moving on: There are a few possible strategies for anticancer therapy based on elevated peroxide, which is a source of oxidative stress but apparently not usually mitochondrially generated. One could simply suppress the excess peroxide, and that’s possible to do experimentally. For example, we work on molecules that suppress ROS, causing a reversing of the transformed phenotype. However, as I’ll show you, other things go on when H2O2 is elevated that might make a reversion of the cancer cell phenotype difficult. One could, as Paul has suggested, enhance cell killing because these cells are already under massive oxidative stress, potentially disadvantaging tumor cells that contain elevated levels of peroxide. But the cells have learned to accommodate the increased peroxide so, paradoxically, killing them with additional peroxide may be difficult. One potentially very interesting source of selectivity might be using the elevated peroxide as a signal to trigger chemistry selectively in the tumor cells. In cancer chemotherapy, it is usual to talk about safety and efficacy; another way of saying that is selectivity. Compounds used therapeutically typically have off-target effects. If the off-target effects are serious, you will not have efficacy at a dose that the host can tolerate. The significantly elevated level of H2O2 in some tumor cells may provide a mechanism for selective toxicity, by delivering toxins to many cells, but activating them only in the cells that have elevated peroxide. Here is a study (Hagen et al., J. Med. Chem. 2012, 55, 924-934) which has appeared very recently, where this is attempted. It uses a chemistry which has become very popular in the last few years in the medicinal chemistry community in which aryl boronate esters are used as pro-drugs and activated by treatment with hydrogen peroxide, which effectively oxidatively cleaves the carbon–boron bond. In this particular pro-drug, which actually contains two toxins, there is a masked quinone methide which the authors of this article generated to scavenge reducing equivalents by consuming glutathione. However, they did not reference the fact that this is a frank alkylating agent which can also alkylate DNA and pretty much any other molecule with a nucleophilic group that it encounters in the cell. So as a model study to establish proof of principle this is fine, but I suspect that as a drug it would not work well. These workers also chose to generate a ferrocene derivative, which will be toxic intrinsically and also because in the presence of peroxide, it will release free Fe2+/3+, which will react further with peroxide in the Fenton reaction – that is to say it will generate hydroxyl radical. Thus two toxic molecules can be generated, hopefully selectively in tumor cells. This slide shows the chemistry in more detail. It shows the conversion of the boronate ester to the corresponding phenol, the latter of which can then release the quinone methide and ferrocene derivatives. The quinone methide can potentially react with nucleophiles, including cellular reducing agents such as glutathione. The ferrocene, in the presence of O2 or H2O2 would yield Fe ions which, in the presence of peroxide, would yield hydroxyl radical, the latter of which is highly toxic. The authors of this study claim to have achieved selectivity at the level of cytotoxicity to cultured cells. The tumor cell line they used was HL60, a human promyelocytic leukemia cell line; the IC50 value was 9 M, which is not terribly potent. They used cultured fibroblasts as controls and didn’t observe cytotoxicity up to 100 M concentration, but obviously this audience will recognize that these are very different types of cells – not a very good comparison! At any rate, at a conceptual level, it’s very interesting and may potentially lead to cancer therapeutic agents capable of exploiting a unique property of tumor cells: elevated hydrogen peroxide. The papers I read involved at least a dozen cell lines, all of which were claimed to have elevated hydrogen peroxide. That said, I do not know how general the phenomenon may be.
One thing that can be done with mitochondria that speaks to the issue of selectivity, and that is often very hard to do in other cells and with other diseases, is to achieve selectivity at the level of delivery to the organelle. This is related to having positive charge associated with lipophilic (or aromatic) groups. If you have them both, you will likely achieve delivery to the mitochondria. This slide shows what is possibly the earliest example – this molecule was first described more than 30 years ago by Jean-Bernard Le Peq and his coworkers. They prepared ditercalinium by making a dimer of an intercalator, i.e. a molecule known to bind tightly to duplex DNA by stacking between base pairs. Ditercalinium was anticipated to bind more tightly to DNA than its constituent monomers, which turned out to work well for this and other bis-intercalators. When ditercalinium was tested in cultured cells, it was found to be stunningly cytotoxic. At the time, the fact that such molecules would be taken up by the mitochondria was not known, so it was a number of years before the actual cytotoxic mechanism was fully understood. This involves binding tightly to mitochondrial DNA after accumulating in the mitochondria, followed by an abortive attempt at repair, which essentially destroys the mitochondrial genome. So ditercalinium provides a splendid example of very highly selective delivery followed by strong cytotoxicity. The slide shows a second molecule that is delivered at high concentration to the mitochondria, namely mitoQ. This series of compounds was originally described by Michael Murphy and evaluated clinically by Antipodean Pharmaceuticals. To my understanding, the clinical candidate failed for reasons of toxicity. Murphy has shown that this compound accumulates in the mitochondrion to a level ~ 500 times greater than the level outside the mitochondrion.

Sidney Hecht: Our studies with the mitoQs suggests that there may be a second problem with these molecules. If they accept an electron (to form the semiquinone radical), they will bear a negative charge on one end of the molecule and a positive charge on the other, linked by a flexible tether. It seems likely that this zwitterion will choose to compensate these opposite charges by positioning them close together spatially. If that happens, it seems unlikely that the molecule can be readily oxidized or reduced, as is the coenzyme Q cofactor that the mitoQs were designed to mimic. In fact, the behavior of the mitoQs in cyclic voltammetry experiments seems to support this hypothesis.

Comment: But the idea, I think, was to generate an antioxidant?

Sidney Hecht: Correct. When I read the papers that deal with these molecules, there is a very careful accounting of the concentrations of the compounds and their accumulation in the mitochondria. To my recollection, the issue of metabolic excretion was less prominent in the papers I have read; the compounds clearly accumulate in the mitochondria, but I am less certain whether the clearance mechanism has been established. Obviously if there isn’t a suitable clearance mechanism, and the compounds simply accumulate in the mitochondria , one might expect to eventually observe cytotoxicity.
There are other examples of drugs delivered to the mitochondrion and the one shown in this study is quite interesting. It is essentially a peptidomimetic, a mimetic for oligoarginine, which has been used as a cell delivery vehicle. This compound incorporates the guanidinium side chain of arginine into a bicyclic structure, and employs four of them in this particular experiment. The compound of interest is attached to a dye so its cellular trafficking into a cell can be monitored. There are two molecules that I’ve highlighted here, one of which has an OH group on this end and one of which has a tert-butyldiphenylsilyl group, which is to say another very lipophilic group at the end of a charged molecule. These can be delivered to the mitochondria of HeLa cells. This is the compound with the tert-butyldiphenylsilyl group at the end and it is internalized significantly better than the compound that doesn’t have this lipophilic group on the end, and also significantly better than cell penetrating peptides having the same fluorophore. Unsurprisingly, for the compound that penetrates better at even 10 micromolar concentration, the toxicity is significantly higher, so if you are going to use compounds like this, you have to recognize that you are accumulating compound in the mitochondria and unless there is someway for it to get out of the mitochondria you have potential trouble. This shows the kinetics of uptake. Within an hour or two there is substantial uptake of the molecule into HeLa cells and this shows that it tracks with a MitoTracker dye and actually is going into the mitochondria. So you can deliver compounds to the mitochondria if you have a reason for doing it. That is a huge advantage relative to the strategy for creating a clinical agent for many other types of therapeutic applications.

So let’s consider the sensing of hydrogen peroxide in mammalian systems.
Here is the first of two papers that speaks to coupling to proteins that are intended to do other things if one has elevated hydrogen peroxide. This paper looked at the ability of molecules to respond to H2O2 as a signaling molecule rather than as a source of redox stress; that is to act as sensors. It is an interesting paper because it compares the transformation of certain proteins that have reactive groups in the the presence of a very large amount of H2O2, a level comparable to those found in cancer cells, but also in the presence of significant (i.e. millimolar) levels of glutathione. At the surface, one might think that one wouldn’t observe any reactivity of the proteins in the cells despite the elevated H2O2 levels because of the huge pool of glutathione available to react with the H2O2 and inactivate it. But there is also a kinetic parameter. There are proteins having amino acid residues that react with H2O2 much faster than does glutathione. So the sensing involves proteins that react with H2O2 faster that it can react with glutathione or with the detoxification catalysts. Enzymes that process H2O2, such as peroxidases and catalase, turn out to have a variety of functional groups that bind to the H2O2 and transform it. In contrast, the molecules that are modified by H2O2 are not there to process it; they are the sensors for H2O2, and almost exclusively share a single type of functional group. They typically have a cysteine, which is an acidic cysteine because the group(s) surrounding it spatially facilitate deprotonation, readily forming a cysteine thiolate, the latter of which is very highly reactive with H2O2 and forms a sulfenic acid. Protein modification occurs in the physiologic H2O2 range, 1 – 700 nanomolar concentration, below the levels typically associated with redox stress. So this is what we were talking about earlier; H2O2 as a signaling molecule, not a source of redox stress. This is the reaction. The sulfur anion reacts with H2O2 to form a sulfenic acid which then reacts with a nucleophile that’s somewhere close by spatially in the protein, forming a covalent bond. Typically this may be another sulfhydryl group, but actually in protein tyrosine phosphatases – it’s a backbone atom that actually forms a linkage to give an S-N bond.

This slide actually shows the physiologic range of H2O2 that can be involved. This is a bacterial oxygen sensor, OxyR, which is transformed quite readily by H2O2. The slide also shows that many proteins require elevated levels of H2O2 to be transformed to the cysteine sulfenic acid and then on to other products in the presence of elevated levels of H2O2. There are mammalian proteins that fall in this physiologic range which will be transformed in cancer cells (having elevated levels of H2O2) but not necessarily in normal cells.

The next article continues this line of thinking. It deals with oxidant signals and oxidative stress. It discusses cellular proteins of interest in aging and diseases under redox control. I’m going to highlight four of the large number of proteins that were talked about. First there is the NADPH oxidase homologue Nox 1. Its overexpression can result in a transformed phenotype in NIH 3T3 cells. The overexpression of the protein increases the basal level of peroxide ten fold and a sustained increase in H2O2 in these cells is essential to maintain the transformed phenotype. The second protein of interest is apoptosis inducing factor (AIF). Apoptosis inducing factor, present in the mitochondria, normally assists in radical and peroxide scavenging, while its translocation outside the mitochondria can trigger cell death and its deficiency is associated with neurodegeneration. So this represents a second interesting protein that is transformed in the presence of H2O2.

There are also a number of phosphatases under redox control, and this now brings us into the realm of the coupling of redox stress to normal signaling mechanisms. Cellular phosphatases include protein tyrosine phosphatases (PTPs) which contain active site cysteines that are easily oxidized to sulfenic acids, especially at elevated levels of peroxide. Cysteine oxidation can alter the balance between the kinase and phosphatase activities. So this is no different than altering the balance to a more phosphorylated or less phosphorylated protein that is involved in signal transduction. But it is doing so not directly through a phosphatase or kinase activity, but by regulation through redox stress.

We will next consider p66Shc: mice lacking this protein have an increased life span. Cells from p66Shc-/- mice have reduced ROS levels. For the protein c-Myc, it was found that forced expression of c-Myc raised ROS levels. This was accompanied by a decrease in SOD2 levels and an increase in peroxiredoxin 3. So the message in this paper is that proteins critical for phosphorylation and dephosphorylation are themselves affected by oxidation and potentially by re-reduction back to the native proteins. The state of the reactive cysteine, by affecting the phosphatase activities, effectively changes the balance between phosphorylated and dephosphorylated proteins. So it’s the same as regulating phosphorylation; but it regulates phosphorylation by regulating redox stress within the cell. This is the model that was suggested in this last paper; at the center of the hub is reactive oxygen species which, through metabolic parameters and the oxygen scavenging and oxidase activities, can affect the balance between senescence or apoptosis and tumor formation. So this offers a slightly different perspective on the linkage between reductive and oxidative stress and tumorigenesis.

Paul Davies: And is it understood how activating an oncogene can elevate levels of ROS?

Sidney Hecht: If it is, it wasn’t described in this paper.

Paul Davies: A simple question to pay attention to: How does switching on an oncogene switch on reactive oxygen species?

General discussion.

Mitochondria and Cancer March 22-23 2012 Final Agenda
Mitchondria, metabolism, and cancer Participant List RMR

2 Responses to “ASU PSOC Workshop, Wednesday March 21st – Friday March 23rd 2012”

  1. […] you want to go directly to the sources, check out this interview with Seyfried at a conference funded by the National Cancer Institute and this interview, which you must watch til […]

  2. […] the ketogenic diet one big step further. He advocates that patients fast periodically. There’s nothing more powerful than calorie restriction in reducing a tumor’s ability to grow blood vessels and spread, he […]

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