Tuesday, December 4, 2012

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.

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