SeeMyScience: Why is the Muscle Gone? Altered pathways in Cancer Cachexia.

Cachexia is a complex condition of muscle wasting affecting about half of all cancer patients. In the last installment of “See My Science”, we discussed what cachexia is, and how it affects patients. In this post, I’ll expand on some of the key pathway changes we focus on, and how they lead to muscle loss.

 

Antioxidants are the enzymes keeping destructive
Free Radicals under control. Source: Doctor Fun

Stressed Out

The body is fighting a constant battle to maintain the status-quo, a fine balancing act where the smallest change in one area could have huge effects down the track. One of these on-going fights is between free radicals and antioxidants. Free radicals are a normal product of bodily function, and are used in the day-to-day functioning of cell systems. However, if overproduced, and not kept in check, these highly reactive species can become seriously destructive, causing damage to DNA, proteins, and other parts of the cell, a state known as “Oxidative Stress”.

Of all the free radical producing enzymes, I’m most interested in one called Xanthine Oxidase (XO). XO is responsible for the conversion of purines, and makes the free radical superoxide (O₂-) as a byproduct. Both superoxide and the end-products of purine conversion help drive the cells in the direction of protein breakdown.

What keeps free radicals in check? Antioxidants. You may know of antioxidants as something you get from heavily hyped “super foods”, but our body actually already has antioxidants in place, their sole purpose in life being to take highly reactive free radicals, break them down (or rather, build them up by donating elections) into less destructive molecules. For example, superoxide can be broken down into hydrogen peroxide and oxygen by SOD, and the hydrogen peroxide can be further broken down into oxygen and water by Catalase and GPx.

My boss explains it in a great way: think of free radicals like a rowdy football team on their end of season trip. They’ve had a few drinks, things have gotten out of hand, and they’re starting to get a bit rowdy. The second they are off the bus, things get a bit rough, and maybe they start breaking a few things, getting into fights. Antioxidants are the bus and the security guards: they keep them contained, and limit the amount they can damage.

When there aren’t enough antioxidants, or they’re not working efficiently enough to mop up the excess free radicals, we start to see the accumulation of damage we associate with oxidative stress. Oxidative stress is known to be a key player in many conditions, including cachexia.

Figure 1. An example of muscle wasting pathways in cachexia. A complex cascade of reactions is triggered by molecules released by the tumour. Source: Vaughan et al 2012, J Cachexia Sarcopenia Muscle.

 

How is Oxidative Stress involved in Cancer Cachexia?

Levels of free radicals and the damage they cause have previously been shown to increase in patients with cancer and cachexia. Several studies have also shown that antioxidants are present in lower levels, or are not working as efficiently in cachexia as they would in a weight-stable or healthy person. This means that they are not able to compensate for the increased activity of free radical-producing pathways, resulting in oxidative stress.

In addition to the damage they cause, free radicals play an important role in cell signaling, where they are involved in the pathways that drive muscle break-down. An example of this is the key muscle-degrading pathway we are interested in, the Ubiquitin Proteolytic Pathway (UPP; See Figure 1).

The Ubiquitin Proteolytic Pathway

The UPP is basically a system that uses ubiquitin molecules to tag proteins that are to be broken down. Ubiquitin is activated by an E1 enzyme, moves to a carrier protein (E2), which then recognizes E3 protein ligases. Two E3s in particular, known as MuRF1 (Muscle RING-finger protein-1) and Atrogin1/MAFbx (Muscle Atrophy F-box), have been suggested as key to the loss of muscle in wasting. Together, the E2 and E3 enzymes attach a chain of ubiquitin enzymes to the target protein (in our case a muscle protein), which can then be unfolded and broken down by a unit called a ‘proteasome’. The proteasome breaks the protein into its smaller building blocks, peptides, which then get broken up into even smaller bits, amino acids. This sudden surge of amino acids can be used to make proteins that further drive the UPP, creating a vicious cycle of muscle destruction, as shown in the bottom section of Figure 1.

Certain components of the UPP are increased in experimental models & patients with cachexia, which means increased potential for them to be tagging our muscle proteins. Increased levels of free radicals can help drive the reactions that lead to the activation of the UPP, by signaling for the control of rate at which the various components are produced.

How are we looking at these pathways?

In order to determine whether there is an increase in potential free radical production in cachectic muscle, and an increase in damage caused by them, we look at the expression levels of genes for our two free radical-producing enzymes of interest,XO and NOX. We also look at the activity of XO, as an increase will indicate that more free radicals are being produced as a by-product. To identify damage caused by free radicals, we look at a marker referred to as 8-OH-dG, which is produced when free radicals damage DNA. We also check what the levels of gene expression are like for certain components of the UPP, as increased expression gives an indication that there is greater potential for the breakdown of proteins by that method.

To figure out what’s going on with antioxidants, we do similar work. I look at the gene expression levels of the different SODs, GPx, and Catalase, and their activity levels in muscle samples. If there is no change in their levels or activity, but there is an increase in free radical production, it means the body is not compensating for the increased potential for damage.  If they have decreased compared to healthy or weight-stable tissue, it means that the there is a decreased ability for the cell to clean up the excess free radicals. Either way, oxidative stress is on the cards.

For this particular study, we were more interested in whether these pathways can be returned to normal (or at very least cleaned up after!) by our two treatments, fish oil and oxypurinol. In my next post, I’ll explain why I think these treatments will be beneficial to cancer patients, and how they fit into the above systems.

Neysa

“See My Science” aims to explain the science done by our group in a manner accessible to the public. The current series focuses on the following publication: Vaughan VC et al (2012) Eicosapentaenoic Acid and Oxypurinol in the Treatment of Muscle Wasting in a Mouse Model of Cancer Cachexia. PLoS ONE 7(9): e45900. doi:10.1371/journal.pone.0045900

SeeMyScience: What is Cancer Cachexia?

When I completed my Bachelor of Science, I was looking for a lab in which to complete my one-year honours qualification. Dr Lewandowski had been one of my lecturers during my undergrad, and his group had started working on an serious condition of muscle loss experienced by many chronic disease patients, called ‘cachexia’. In Part 2 of "See My Science", I'll begin to explain what cachexia is, and why researching it is important.

Cachexia, affecting 50% of cancer patients, causes extreme loss of muscle and fat (Source)

 

What is Cachexia?

Cancer Cachexia (pronounced car-kex-ear) is the unintentional loss of muscle and fat that occurs in many patients with cancer. While cachexia is seen in several other diseases, such as HIV/AIDS, sepsis, chronic obstructive pulmonary disease and congestive heart failure, the loss of muscle has been shown to occur most rapidly in cancer patients. 

Patients are said to have “[…] a complex metabolic profile […]”, meaning that there are many changes in their metabolism which are causing these issues, resulting in more energy being used by the body than is being put in/stored. This also means that the metabolism of muscle is altered, i.e. the rate of muscle break-down is faster than the rate of creation of new muscle. Part of this is in response to the increased energy demand (the body can break down muscle in order to obtain more energy), but it is also caused by complex chain reactions  triggered by the body’s response to the tumour. This is just one example of a pathway involved.

Patients with cachexia have a lower quality of life than patients who do not lose weight, experiencing greater pain, fatigue, and reduced mobility as muscles become less functional. Weight-loss also reduces the effectiveness of chemotherapy, with cachectic patients having to have lower doses, for shorter periods, and often unable to undergo as many rounds of therapy. Developing an effective treatment for cachexia would help cancer patients to have a better quality of life, and also help improve their chances of successful therapy.

The most common definition used at present is involuntary weight loss of greater than 5% from historical weight, a body mass index (BMI) less than 20 with any degree of weight loss greater than 2%. However, these measures only take into account weight, ignoring the vast number of other serious symptoms. Therefore, newer definitions are supported by other symptoms, including inflammation, fatigue, and decreased quality of life. See Fearon et al 2011 for the consensus definition.

How common is Cachexia?

It is generally thought that about half of all cancer patients will develop cachexia, although this can rise to as much as 80% in the later stages of cancer. In is most common in cancers of the pancreas, colon and lung. 45% of patients with cachexia lose more than 10% of their original body weight, and patients who lose 30% of their original body weight will unfortunately usually succumb, with around 20% of cancer-related deaths thought to be attributable to cachexia.

Despite how common cachexia is, it is often underdiagnosed. This may be because weight-loss is seen as an inevitable part of cancer. Sometimes it is also because the tumour itself it considered more urgent than the loss of weight. Weight-loss is also often thought to be a side-effect of cancer therapy, and while this is true to a certain extent, many cancer patients lose weight prior to undergoing chemo or radiation, or even before they receive a diagnosis.

Why don’t they just eat more?

Unlike some forms of weight-loss, simply eating more does not cure cachexia. Neither does smoking pot, sorry. Improving nutrition is vitally important to the treatment of cachexia (you can’t make muscle out of thin air!), but alone, it is not enough, because reduced calorie intake is not the underlying cause. This is because of those complex pathways I mentioned before, which are telling the body to break down muscle and fat. We need to stop the processes that cause the body to break down muscle, but in the past, this has proven to be very difficult due to the complexity of the condition.

I like to think of it like a busy city. You might have a normal route you take from your house to work. One day, a tree falls across the road, and you can’t get by. What do you do? Back-track, and take another route! The body can to the same thing: you stop one pathway, only for another to compensate. The only way you’re not getting to work is if every street, railroad and back alley is impassable. In order to stop cachexia, we have to hit it in lots of different places. This is called a multi-target approach, which I will discuss in a few post’s time, but generally, it will involve improving nutrition, targeted exercise, and a combination of pharmaceuticals that will inhibit some things and promote others.

At present, we do not have a globally effective treatment for cancer cachexia. There are some treatments that may work for some people, but not others. The aim of our study was to look at two potential treatments, both alone and in combination, to figure out whether they were able to slow down, or even stop, the loss of muscle we see in cachexia.

Next post, I’ll be explaining the pathways my research focuses on, and how they interact with the body. In the mean-time, is there anything you would like to know about cachexia that I haven’t covered here? I’d love to hear from you, so either leave a message below, or drop me a line.

Neysa.

“See My Science” aims to explain the science done by our group in a manner accessible to the public. The current series focuses on the following publication: Vaughan VC et al (2012) Eicosapentaenoic Acid and Oxypurinol in the Treatment of Muscle Wasting in a Mouse Model of Cancer Cachexia. PLoS ONE 7(9): e45900. doi:10.1371/journal.pone.0045900

SeeMyScience: On the Topic of Animal Testing

Earlier this year, I had the opportunity to participate in the “I’m A Scientist, Get me out of here” program, in which students from around Australia ask a variety of scientists questions about every topic imaginable, from what the universe is made of, to what subjects we studied at school. We had some fantastic questions, including one in particular that highlighted one of the most controversial issues in medical science: 

“What do you feel about using animals in experiments and testing experiments on them?”

Animal testing is a loaded topic, and not often spoken about, because scientists often fear the backlash that comes from people who do not understand or approve of what it is we do. As part of this series, I felt it would be appropriate to reproduce my response to this important question, in order to help explain what goes into the development of treatments for some of our most debilitating diseases.

"[This is a question] that is discussed a lot by scientists, particularly in medical research. This is going to be a very long answer, but it is a very big issue! First up, I will be honest: I agree with animal testing in medical research, but not for cosmetics. Now, let’s go in to a bit more detail about why I have these opinions. Let’s start with medical research. I’m going to use drug development as an example, because it’s what I’m familiar with, but this could apply to any disease.

"When a scientist comes up with a new idea for a drug that helps treat cancer, we can’t just take that drug and immediately start using it on people. We don’t know what dose to use, if it will actually cause more harm than not taking the drug, if it will make the cancer get larger, how it interacts with all of our organs. Some new drugs might cause people to have heart failure, or injure their brain, cause birth defects, or just make them feel really sad. In extreme cases, they might even die, so as you can see, we don’t want to go testing things on people straight away. So, where do we start? Hint: It’s not actually with animals!

"Once we’ve done lots of reading about this compound, and figured out how we think it will work, we can do things like run it through a computer simulation. These programs can be quite complex, and give you lots of data about how the body MIGHT react to the drug. This step can rule out lots of drugs, when the computer reminds the scientist that Drug X actually acts on nerve Y in a bad way, so it gets ditched. Even if our drug passes this step, the computer doesn’t know everything about how the human body works (even we don’t!), or how the drug might react with environmental factors. So, after we have gained insight from our computer program, we take it to cell models.

"There are all different kinds of cells we grow in the lab, which can represent all different tissues and diseases. The first thing we do is figure out how much of our drug will cause the cells to die. We then work backwards, using lots of different dilutions to figure out the minimum amount of drug we need to have an effect on the cells. Think of it like making up cordial. We know that straight cordial doesn’t taste very good, but not enough cordial just tastes like water, so we want to get just the right amount to taste good! Once we find this level, we can study the cells to see how they react to the drug. This might be changes in the way they look, what genes they are producing, how well their enzymes are working. We can make sure it is having the effect on the cells we think it is supposed to, or figure out why it is having ones we don’t know about. We do all the same tests in different types of cells, to see what effect it will have on different tissues. This step can take a very long time, and even more drugs never make it past this stage, because they simply do the wrong thing, or are too toxic.

"So, our drug has passed cell culture testing, we think it’s pretty safe, we know how much we need to use, and we’re ready to stick it into a human! NO WAY! We know it’s not killing off the cells in culture, but those are just cells. The body is made up of organs, and blood, and enzymes, all of which can take a drug and change it. This is called bio-metabolism, and some of the products can be quite poisonous. Unfortunately, cell models can only recreate these conditions to a small extent, and we certainly can’t replicate our complex organs in a petri-dish. Without knowing what it will do when it’s taken into our livers, kidneys, brains, etc., wouldn’t you rather have more information? This is where animals come in.

"The only way we can know how a drug will react in complex systems is to place it into a complex system. We want it to be comparable to humans, so we need an animal that has similar genes, organs, and biological processes to us, which can have the same diseases as humans have. In many cases, the closest animals are mice. I’ve already talked a little bit about how mice relate to the human body here.

"Before we work with animals, we have to be very sure that our drug is going to work, and that it is going to be safe. We have to do a lot of math to make sure that we use as few animals as possible, but enough that our results will be meaningful. Even before we see the animals, we have to talk to other scientists, vets, and people from the community, to figure out if this research is going to help people, if we are doing the right thing, a process known as ethics approval. We have to make sure that our animals are going to be under as little stress as possible, that we can reduce stress if we have to, and that we are not going to put the animals in pain. Only after we are very sure of all these things do we ever test on animals.

"When we are running animal trials, we check our animals every day, sometimes twice a day! We make sure they have enough food and water, that they are socialising with other animals, and that they are comfortable. If we ever see that they are in pain, or stressed, we do everything we can to help the animal. Sometimes we have to give them an injection, or take some blood, just like going to the doctor for a blood test. When we give them our new drug, we keep a close eye on them, and if it looks like it’s having a bad effect, or an effect we weren’t expecting, we stop straight away. And at the end of the study, we put the animals to sleep quietly, so that they don’t feel any pain. Some people are lucky, and don’t have to give their animals drugs. Some people give them different food, and Emma from the Organs Zone gets them to run through mazes to check their memory, or listens to them sing to each other!

"The life of the animals we use in research gives us valuable information that can help us improve or save many human lives. It can be very difficult sometimes, and sad, but we treat the animals with respect, and I thank each for the contribution they are making. We do not let anything go to waste, looking at all of the organs and other tissues, and keeping all the parts we don’t use so that other people can look at the same drug or disease without having to do the experiment all over again with new animals.

"I don’t agree with animal testing of cosmetics for reasons that are linked to those I’ve already spoken about above. A new type of hair dye or eye shadow isn’t going to save lives, or really even make them better. We already know so much about the ingredients in products we use on our bodies, that I don’t see why we should use more animals to test them."

I often speak to people, even within my own family, who are against animal testing. Each has their own particular reason, and discussions can become quite heated. Scientists are often told they are evil, amoral, their lives sometimes even threatened, because we have to use these methods in order to complete our research. We can try to reason and explain, but many of us do not, placing it in the 'too hard' basket. I do not enjoy testing on animals, but I know that in doing so, we may be able to help many people in the future.

Neysa.

SeeMyScience: Explaining Our Published Papers

"You do what now?"

As a scientist, the most frequent comment I get when I tell people what I do is some variation of “I have no idea what that means, but it sounds important”. This was perfectly illustrated recently, in response to a link I posted on Facebook about a paper we published (Pictured Left).

Let’s be honest, many scientists suck at talking about their work in a way that people who are not scientists can understand. Part of this is the way we are trained to write: technical papers for science journal publication. However, as I said in a recent blog post, we need to do better. We need to talk about our science in a way everyone can understand. And it’s about time I put my money where my mouth is.

In the coming weeks, I will be posting a series that looks at the paper our lab group recently published about our work developing a treatment for Cancer Cachexia. In this series, I will be combing through the article, explaining what we do, why we do it, what we learn from the results, and how we decide what to do in the future.

EPA and Oxypurinol Treatment of Cancer Cachexia

• On the Topic of Animal Testing
• What is Cancer Cachexia?
• Why is the Muscle Gone? Altered Pathways in Cancer Cachexia
• Fighting Back - Why EPA & Oxypurinol?
• Growing Cachexia-Causing Cancer Cells
• Explaining treatment groups and ‘interventions
• Weight-loss and Muscle Mass
• What happened with Oxidative Stress?
• Was muscle mass being controlled?
• Putting it All Together
• Where to now?

“See My Science” aims to explain the science done by our group in a manner accessible to the public. The current series focuses on Vaughan VC et al (2012) Eicosapentaenoic Acid and Oxypurinol in the Treatment of Muscle Wasting in a Mouse Model of Cancer Cachexia. PLoS ONE 7(9): e45900. doi:10.1371/journal.pone.0045900