Efficacy of Omeg-3 Fatty Acids in Preventing Cancer Induced Cachexia

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Efficacy of Omega-3 Polyunsaturated Fatty Acids for Preventing Cancer-Induced

Cachexia

Josh Nooner, BS, CSCS

NSCI – 5843

2/29/16

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IntroductionCachexia is an extremely debilitating condition that is seen in many chronic and terminal

diseases such as cancer. This syndrome is characterized by unintentional weight loss, anorexia,

and severe muscle wasting. Stewart reports, “20% of all cancer deaths are caused directly by

cachexia (Stewart, 2006).” As the cause of death for 1 in 5 cancer patients, an individual who

develops this wasting syndrome has a very poor prognosis. In fact, “Up to 50% of cancer patients

suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia,

resulting in weight loss, a reduced quality of life, and a shortened survival time (Tisdale, 2009).”

As we can see, cachexia is a very prevalent condition, affecting half of all individuals diagnosed

with cancer and is the direct cause of death for 20% of cancer deaths. Additionally, this

syndrome results in a lower quality of life and a reduced survival time, making it an extremely

important target for therapy. Cachexia is currently being heavily studied, as we aim to find an

effective intervention strategy that will reduce the occurrence of this devastating syndrome,

increase the quality of patient’s life’s, and lengthen their survival time. Omega-3 polyunsaturated

fatty acids (PUFAs) are one area of research that is being investigated for their use in alleviating

this wasting syndrome, specifically through their anti-inflammatory properties and their ability to

mediate specific metabolic processes that are seen in cachexia. After studying the altered

metabolic pathways that present in cachexia, we can better understand how the omega-3 fatty

acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), work to alleviate cancer-

induced cachexia.

In order to better understand this syndrome, we must first define exactly what it is. In 2011

Fearon brought together an international panel of experts that formed a consensus on the

definition, diagnosis, and the classification of cachexia. They reported that, “Cancer cachexia is

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defined as a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass

(with or without loss of fat mass) that cannot be fully reversed by conventional nutritional

support and leads to progressive functional impairment (Fearon, 2011).” This definition brings

up two very important characteristics of cachexia that are crucial to understand. First, cachexia

cannot be fully reversed by conventional nutritional support. Unlike with starvation induced

weight loss, simply adding calories into the diet does not reverse cachexia. Secondly, cachexia

leads to progressive functional impairment. The longer that someone is in a cachectic state, the

more functionally impaired they become, eventually losing all strength which is often the cause

of death. Knowing this helps us to understand why practitioners need to actively manage this

condition and why it is such an important area of research.

We know that cachexia occurs along a spectrum with three stages of severity, and that

there are specific clinical aspects of each stage. The three stages are precachexia, cachexia, and

refractory cachexia. The main clinical characteristics of cachexia are weight loss, anorexia or

loss of appetite, reduced food intake, complex metabolic changes, systemic inflammation, and

reduced functional capacity. There is a stream of negative side effects, each one causing another,

with each further step downward compounding the entire cycle. This is an extremely viscous

catabolic cycle that is repeated over and over eventually leading to death in the majority of

cancer patients.

The metabolic processes seen in cachexia are quite complex, as the cellular mechanisms are

not typically seen in normal healthy cells, or in cells of those experiencing starvation induced

weight loss. Barber reports, “The fundamental difference between the weight loss observed in

cachexia and that seen in, e.g., starvation, is the lack of reversibility with feeding (Barber,

2001).” Simply adding calories to the diet does not reverse weight loss seen in cachexia. These

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metabolic alterations are thought to be induced by a combination of both tumor and host

changes. Barber continues and states, “Candidate mediators include cytokines, neuroendocrine

hormones, and tumor specific products. Several pro-inflammatory cytokines, including tumor

necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and interferon-y, have been

implicated in cachexia (Barber, 2001).” Chronic inflammation is one of the major clinical

manifestations of cancer, resulting in an elevation of pro-inflammatory cytokines, which mediate

cachexia.

Hypermetabolism is another complication of cachexia that leads to muscle and fat loss.

“Approximately 50% of patients are hypermetabolic (REE > 110% of predicted) …

hypermetabolism has been related to an elevated adrenergic state or systemic inflammation

(Fearon, 2012).” Cachectic patients are in a hypermetabolic state, burning more calories than

they would under normal conditions, yet have very limited appetites, thus they are also

consuming fewer calories than they would under normal conditions. The hypermetabolic state is

thought to be caused, in part, by futile cycles. Futile cycles are when opposing metabolic cycles

(glycolysis and gluconeogenesis) occur simultaneously, resulting in the use of ATP and

production of heat.

Tumor cells are known to have an increased uptake of glucose, even in the presence of

adequate oxygen levels. This phenomenon, known as the Warburg effect, causes excess

production of lactate and upregulates the Cori cycle. Fearon continues, “Increased Cori cycle

activity has been documented in weight-losing cancer patients… Overall glucose flux has been

shown to be increased in weight-losing cancer patients, and such flux has been estimated to

contribute up to 40% of the increase in energy expenditure in metastatic cancer (Fearon, 2012).”

Here Fearon describes one of the main causes of hypermetabolism seen in cachexia. The second

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known cause of increased metabolism is a heightened expression of mitochondrial uncoupling

proteins (UCPs). This causes, as Fearon explains, “Dissipation of the proton gradient so that

oxygen consumption is no longer coupled to ATP synthesis and heat is generated (Fearon,

2012).” This effect is commonly seen in the brown adipose tissue (BAT) of cachectic cancer

patients, causing a thermogenic effect. The combination of an increased glucose flux and an

increased expression of UCPs in adipose tissue creates a rise in heat production. These are the

two main causes of hypermetabolism seen in cancer-induced cachexia.

Lastly, cachexia is characterized by alterations in gene expression and protein levels. As

previously stated, there is an overexpression of mitochondrial uncoupling proteins. This effect

increases heat production, ATP loss, and creates a hypermetabolic effect. Another gene that is

altered is the GLUT-4 receptor protein gene. GLUT-4 expression is downregulated in cachexia

and causes insulin insensitivity. Reductions in insulin sensitivity create a pro-catabolic state,

leading to increased use of the Cori cycle and loss of muscle mass.

Additionally, both muscle and adipose tissue loss are due to expression of specific proteins

seen in cachexia. Tisdale describes, “Loss of adipose tissue is due to increased lipolysis by tumor

or host products. Loss of skeletal muscle in cachexia results from a depression in protein

synthesis combined with an increase in protein degradation. The increase in protein degradation

may include increased activity of the ubiquitin-proteasome pathway (Tisdale, 2009).” Tisdale

explains that loss of adipose tissue is due to lipolysis brought about by certain factors. One such

proposed factor is zinc alpha2-glycoprotein (ZAG) which was discovered to be a lipid

mobilizing factor. This protein has been shown to cause adipose tissue loss in many animal and

human cell studies. Tisdale concludes, “An increased ZAG expression may be responsible for

the increased lipolytic response of adipose tissue in cancer cachexia (Tisdale, 2009).” Mimicking

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this fat wasting protein is a muscle wasting glycoprotein called Proteolysis-Inducing Factor

(PIF). PIF has been found to induce protein degradation and prevent protein synthesis in

numerous animal, cell, and human models of cancer cachexia. Furthermore, the ubiquitin-

proteasome pathway is highly active in cachexia, causing the majority of muscle loss. Jatoi

explains that, “The ubiquitin-proteasome pathway is responsible for >80% of lean tissue wasting

from cancer (Jatoi, 2005).” A direct correlation exists between the degree of expression of the

ubiquitin-proteasome pathway and how advanced a tumor is. In summary, although not an

extensive list, there are many proteins that are known to be over expressed in cancer-induced

cachexia.

Now that we have a better understanding of the mediators and mechanisms involved in

cachexia we can look at how omega-3 PUFAs act on these pathways to reduce cachexia. There

have been a number of quality papers published detailing the beneficial effects of omega-3

PUFAs on cachexia. In 2005 Jatoi reported that, “The purported mechanisms of n-3 PUFA in

preventing lean tissue wasting are through the suppression of the ubiquitin-proteasome pathway,

inflammatory cytokines, and the cancer cachectic factor (Jatoi, 2005).” As previously discussed,

the ubiquitin-proteasome pathway accounts for a vast majority of muscle loss and the

inflammatory cytokines lead to hypermetabolism.

In 2008 Rondanelli reported, “Production of pro-inflammatory cytokines such as IL-6, IL-1,

and TNF can be downregulated by EPA. Furthermore, the effects of PIF, are also inhibited by

EPA (Rondanelli, 2008).” Rondanelli discusses EPA’s effect on cytokines, which are chronically

elevated in cachexia. Adding to this, Fetterman elaborates, “Supplementation with n-3 PUFA

also appears able to reduce production of proinflammatory cytokines, such as IL-1, IL-6, IL-8,

and TNF-a…Excess activity of these substances contributes to pathological inflammation

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(Fetterman, 2009).” Reduction in pro-inflammatory cytokines is one of the main reported

mechanisms and a major benefit of n-3 supplementation for cachexia patients.

Furthermore, a recent report summarized the antineoplastic activity of n-3 PUFAs. “The

three main antineoplastic activities of n-3 PUFAs that have been proposed are (i) modulation of

COX activity; (ii) alteration of membrane dynamics and cell surface receptor function and (iii)

increased cellular oxidative stress (Cockbain, 2011).” This evidence suggests n-3 PUFAs can

beneficially effect eicosanoid synthesis through COX enzyme modification, can be incorporated

into the lipid bilayer and alter the structure and function of the cell surface and its downstream

signaling pathways, and can also beneficially alter the intracellular redox status of target tissues.

Adding to these beneficial effects, Hardman states, “n-3 fatty acids may be detrimental to

the growth of metastatic or residual cancer cells by altering eicosanoid metabolism, slowing

cancer cell mitosis, increasing cancer cell death, inducing differentiation, suppressing

angiogenesis, and altering estrogen metabolism (Hardman, 2002).” Hardman shows us two more

benefits of n-3 PUFAs that have yet to be discussed, angiogenesis and estrogen. By preventing

vascular supply to the tumor, fish oil deprives the tumor of nutrients needed for growth. By

decreasing estrogen production, n-3 PUFAs prevent the activation of estrogen receptors on

breast, colon, and prostate cancer cells, which all have estrogen receptors needed for growth.

More recently, Murphy reported many more benefits of n-3 PUFAs, stating, “EPA may

support the anabolic potential of muscle through sensitizing skeletal muscle to insulin. EPA has

been shown to improve glucose uptake and increase GLUT-4 expression in skeletal muscle

(Murphy, 2011).” This is a highly desirable effect, noting that muscle protein synthesis must

occur in an anabolic state created by the hormone insulin. Murphy continues, “EPA and DHA

reduced the side effects from chemotherapy, and limited weight loss and anorexia. EPA and

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DHA have also been reported to enhance tumor response to chemotherapy (Murphy, 2011).”

These reports that EPA and DHA can increase the positive effects of chemotherapy while also

reducing the side effects just add to the list of reasons someone would consider taking fish oil to

treat cancer-induced cachexia.

Now that we have reviewed the main pathways through which omega-3 fatty acids inhibit

cancer-induced cachexia, we can take a deeper look into the literature supporting both sides of

the argument. In order to fully understand this complex issue a full literature review is necessary.

The following section will be a review of the major studies that support the use of fish oil for

cancer patients with cachexia. I will then review the major studies that discourage using fish oil

to treat cachexia. Lastly, I will summarize the major findings of these studies, discuss the

strengths and weaknesses of each study, and provide a recommendation for their use.

Review of Supporting Articles The first point of view on this issue that will be discussed is that Omega-3’s are effective

for the treatment of cancer induced cachexia. Beginning with the earliest studies on this issue and

progressing chronologically, the main studies on this subject will be discussed in detail. Included

in the review will be the purpose of each study, the methodology, the results of the study, and the

researcher’s conclusions. The opposing point of view on this issue will be discussed

subsequently, with each study supporting this view point presented chronologically and

discussed in a similar fashion.

Omega-3 PUFAs began to be investigated for their potential ameliorating effects on

cachexia as early as 1975. The study of fish oil began when epidemiological studies, often

studying Greenland Inuits or Native Alaskans, first showed that cultures eating higher amounts

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of fish had lower incidence of chronic diseases such as heart disease, diabetes, and cancer. One

study reported, “Alaskan and Greenland Eskimos have lower rates of colon cancer and a higher

consumption of n-3 fatty acids than other North Americans (Chang, 1998).” This study, among

others, showed that there was a beneficial effect of consuming higher amounts of n-3 PUFAs,

mainly from fish, and kick started the investigation into fish oil. Since then, thousands of studies

have been done, investigating fish oil’s effects on hundreds of clinical outcomes, including

cancer-induced cachexia.

Many studies have been conducted to determine the appropriate dosage of fish oil and to

test for adverse side effects and toxicity. In 1999 a phase 1 clinical trial was conducted to

determine the maximal tolerated dose of fish oil. Patients were given .05g/kg/day fish oil

supplements that were to be increased by .05g/kg/day every 2 weeks unless a complication arose.

The authors found that, “At doses of 0.35 g/kg/day and above, at least one-third of patients

suffered a dose-limiting toxicity. Therfore, the maximum tolerable dose was considered to be 0.3

g/kg/day (Burns, 1999).” Important to note is that the “toxicity” discussed in this study were all

very minimal side effects. Burns explains, “These included excessive belching, fish taste in

mouth, fish taste of food, fish smell on their own body, flatulence, and diarrhea… In summary

we found that patients with advanced cancer can tolerate a large dose of fish oil with only minor

side effects (Burns, 1999).” This study used much larger doses of fish oil than most other studies

and found only minor side effects.

One of the earliest studies that will be discussed was published in 1990 in the Journal of

Cancer Research. The purpose of this study by Tisdale and Dhesi was to determine if omega-3

fatty acids could inhibit weight loss in an experimental cachexia model (Tisdale, 1990). In order

to test this, they transplanted MAC16 tumor fragments into the flanks of NMRI mice to induce

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experimental colon adenocarcinoma. After tumor implantation was confirmed, the mice were

randomized into one of two dietary treatments. “The standard diet was comprised of mouse

breeding nuts which contained 50% carbohydrate, 20% protein, 4.1% crude oil and supplied

11.5% of the energy as fat (Tisdale, 1990).” All other treatment arms were isocaloric but

decreased the carbohydrate amount and provided an increasing amount of calories from fish oil,

from 5% up to 50% of total energy being derived from fish oil. The mice were weighed daily and

remained on the diet for 24 days.

The authors found that, “Weight loss was prevented in proportion to the concentration of

fish oil in the diet with almost complete protection occurring when the fish oil comprised 50% of

the diet (Tisdale, 1990).” Here we can see that the fish oil supplemented groups did show a

reversal of weight loss. The authors also reported that the weight loss inhibition was seen in a

dose dependent manner, with the groups receiving the largest amounts of calories from fish oil

losing the least amount of weight. Secondly, the authors reported, “Both the tumor weight gain

per day and the tumor volume increase were reduced in animals fed the fish oil diets (Tisdale,

1990).” Interestingly, the fish oil not only prevented weight loss, but it also prevented the tumor

from growing as quickly as it did in the control group. This is important to note, because many

studies on fish oil report there being a beneficial effect on both the host and the tumor cells. The

authors conclude by stating, “Diets enriched with fish oil have been shown to reduce both the

growth rate and the extent of weight loss produced by the MAC16 adenocarcinoma…Thus an

EPA-containing oil has been shown to exert both antitumor and anticachectic activity with no

toxicity.” This study was an important early study that shed a hopeful light on the cachexia

dilemma and opened the gateway for more research into the effects of fish oil.

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Five years later, Tisdale was involved in another study with six other researchers that

investigated how polyunsaturated fatty acids affected cachexia progression in a group of patients

with pancreatic cancer (Wigmore, 1996). This study looked at pancreatic cancer patients because

cachexia is highly prevalent in this type of cancer. The authors were specifically looking at the

effect of fish oil supplementation on weight loss and the acute-phase response in these patients. It

has been noted that acute-phase protein elevation, “Is associated with both increased resting

energy expenditure and shortened duration of survival. This elevation is thought to be the result

of proinflammatory cytokine release. (Wigmore, 1996).” The authors measured C-reactive

protein (CRP) levels at baseline and 3 months after intervention to examine the acute-phase

response.

A total of eighteen patients with pancreatic adenocarcinoma were included in this study.

The patients were given fish oil capsules of 1g that contained 18% EPA and 12% DHA. Each

week the dose was increased by 2g/day, starting with 2g and rising to 16g/day. Weight, CRP

levels, and a nutritional assessment were taken at baseline, 1 month after treatment, and 3 months

after treatment initiation. Results of this study showed that, “All patients were losing weight

before supplementation, whereas on receiving fish oil there was a median weight gain of

0.3kg/month (Wigmore, 1996).” There was a significant decrease in weight loss in the majority

of the patients after 3 months of supplementation with fish oil. In addition, “After 1 month of

oral fish oil, patients showed an attenuation of the acute phase response by a reduced C-reactive

protein concentration (Wigmore, 1996).” The change in weight was mirrored by changes in CRP

levels. The authors conclude by stating, “Before supplementation, all of the study group

experienced progressive weigh loss; however, following administration of fish oil three quarters

of the group either become weight stable or actually gained a small amount of weight (Wigmore,

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1996).” This study showed that polyunsaturated fatty acids prevented weight loss in pancreatic

cancer patients with cachexia.

The next study in the review was a prospective randomized control trial that was

published in 1998 by Gogos et al. The aim of this study was to investigate the omega-3 PUFAs

and vitamin E effects on survival rates and the immune system in both malnourished and well-

nourished patients with malignancy (Gogos, 1998). The 60 patients were randomized into two

groups. The first group was supplemented with fish oil, and the second group received a placebo.

There were 15 malnourished and 15 well-nourished patients in each group The groups were

followed until the time of death. Before treatment and on day 40 measurements of T cells, T

helper cells, T-suppressor cells, natural killer cells, and synthesis of interleukin-1 (IL-1),

interleukin-6(IL-6), and tumor necrosis factor (TNF) were taken in blood mononuclear cells

(Gogos, 1998).

The results of this study showed that omega-3 polyunsaturated fatty acids produced an

immunomodulating effect and increased the ratio of T-helper cells to T-suppressor cells in the

malnourished group. However, there were no significant changes in cytokine production between

the groups. In terms of survival, the well-nourished group supplemented with fish oil had the

greatest survival whereas the malnourished group receiving the placebo had the least survival.

Additionally, both the well-nourished and the malnourished groups receiving fish oil showed a

significant increase in survival compared to the placebo groups. The authors conclude by saying

that omega-3 PUFAs could offer support, especially to malnourished patients, due to their unique

anticachectic, antitumor, and immunomodulating effects (Gogos, 1998).

The next study supporting the use of omega-3 PUFAs to treat cancer cachexia was

published in 1999 in The Journal of Nutrition. This study by Barber investigated the use of a

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supplement enriched with fish oil on acute-phase protein concentrations in patients presenting

with pancreatic cancer (Barber, 1999). For three weeks, 18 patients were given a nutritional

supplement (2g EPA/day and 1g DA/day), and an additional 18 patients who did not receive the

supplement were used as a control. The APP concentrations were measured at baseline and again

after three weeks of intervention.

The APP measurements taken at the three-week mark revealed that only transferrin was

increased in the fish oil supplemented group. However, in the control group, there were

significant decreases in albumin, pre-albumin, and transferrin, as well as a significant increase in

CRP levels, suggesting an attenuation of the APP response by fish oil. In summary, the authors

conclude, “Fish oil, in combination with a nutritional supplement, is able to prevent progression

of the APP response and cachexia in weight losing patients with advanced cancer (Barber,

1999).”

The same year as the previous study, Barber published another study looking at the

effects of a fish oil enriched nutritional supplement in those with advanced pancreatic cancer.

The purpose of this study was to determine if weight gain can be produced in pancreatic cancer

patients through the use of a conventional nutrition supplement in combination with EPA

(Barber, 1999). Barber administered nutritional supplements enriched with fish oil to 20 patients

with pancreatic adenocarcinoma. There were asked to consume two cans a day, each can

containing 1.09g EPA, 16.1g of protein, and 310 calories, in addition to their normal daily food

intake. Assessments were taken at 3 and 7 weeks for weight, body composition, dietary intake,

resting energy expenditure, and performance status (Barber, 1999).

The results showed improvements in every area that was being studied. The authors

reported, “Patients had significant weight gain at both 3 and 7 weeks (median 2 kg), dietary

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intake increased significantly by almost 400 kcal/day, resting energy expenditure fell

significantly, and performance status and appetite were significantly improved (Barber, 1999).”

This study was able to show that fish oil not only stopped weight loss, but also lead to

improvements in appetite, increased functional performance, as well as a reduction in resting

energy expenditure, going above and beyond the results of previous studies. The very promising

results from this study lead the authors to conclude that cachexia can be countered through the

use of a nutritional supplement enriched with EPA.

Following this encouraging study, Barber decided to look into some of the metabolic

components of cachexia to see how fish oil affected these pathways. In 2001, he published

another study using the same fish oil enriched nutritional supplement, this time looking at how

the supplement effected metabolic mediators that are known to have a role in cachexia (Barber,

2001). Barber provided a supplement that contained 2g of EPA and 600 kcal to 20 patients with

pancreatic cancer who were to consume it daily. Barber reported that, “At baseline and at 3

weeks, patients were weighed and serum concentration samples were taken to measure IL-6,

cortisol, insulin, leptin, blood mononuclear cell production of IL-1B, IL-6, and TNF-a, and

urinary excretion of proteolysis inducing factor (PIF) (Barber, 2001).”

Interestingly, Barber found that after 3 weeks of fish oil supplementation, levels of IL-6

were significantly decrease, the serum insulin concentration was elevated, the cortisol-to-insulin

ratio decreased, and there was also a reduction in the number of patients excreting PIF in the

urine. Notably, there was also weight gain in addition to the beneficial changes in these

metabolic mediators. In conclusion, Barber stated that the fish oil-enriched supplement

modulates different mediators involved in cachexia catabolism. Normalization of cancer

associated metabolic changes, that normally prevent weight gain, are suggested to be brought

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about by the fish oil in the supplement (Barber, 2001). Here we begin to see some of the

mechanisms by which fish oil exerts its positive anti-cachexia effects, specifically through the

modulation of key metabolic pathways. The last three studies by Barber build upon each other

and present a strong argument for the use of omega-3 fatty acids in preventing cachexia in cancer

patients.

Further strengthening the argument for the use of fish oil to treat cachexia is a study that

was published in 2001 by Hardman. The purpose of this study was to determine if concentrated

fish oil will be a useful adjuvant treatment for chemo, as well as to determine if the concentrated

fish oil would cause detrimental increases in the drug toxicity to either the host or tumor cell

(Hardman, 2001). The methods of this study used MDA-MB 231 breast cancer cell xenografts

and implanted them into 120 athymic mice. The mice were fed a normal diet for three weeks to

allow the tumor to develop. At the three-week mark, the mice were randomly divided into two

dietary groups. The first group receiving the standard diet of 5% corn oil, and the second group

receiving a diet containing 3% fish oil concentrate and 2% corn oil. The mice were kept on their

respective diets for two weeks at which point mice from each group were randomly selected to

receive doxorubicin (DOX) therapy for a total of five weeks. Tumor size was measured weekly.

The data from the results of this experiment showed that the 3% FOC group increased the

efficacy of doxorubicin against tumor growth without causing an increase in toxicity to the host

cells. Additionally, “Consumption of the 3% FOC alone was as effective against the growth of

the MDA-MB 231 breast carcinoma as was DOX chemotherapy with the 5% corn oil diet

(Hardman, 2001).” These results are intriguing, showing that a fish oil concentrate increased the

effectiveness of doxorubicin chemotherapy without causing toxicity to the host. Even more

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interesting, this study showed that fish oil concentrate alone was just as effective as

chemotherapy in preventing tumor growth in breast cancer xenografts.

The next study to be reviewed looked at the use of fish oil to treat colorectal cancer. In

2003, Llor published a study in the Journal of Clinical Nutrition that looked at how fish oil

affects colorectal metabolic processes in comparison to olive oil, linoleic acid, and oleic acid

(Llor, 2003). The purpose of this study was to determine the function of these fats in the process

of colorectal carcinogenesis. The authors used both Caco-2 and HT-29 colon adenocarcinoma

cell lines. These cells were then supplemented with fish oil (50% EPA and DHA), olive oil, oleic

acid, or linoleic acid. At 48 and 72 hours after the addition of the fats, the cells were tested for

apoptosis, proliferation, differentiation, and Cox-2 and Bcl-2 expression was assessed.

The results showed that both fish oil and olive oil increased the induction of apoptosis in

both colon cancer cell lines. Fish oil supplementation drastically decreased cell proliferation in

the Caco-2 and HT-29 cell lines. All four of the fats increased cellular differentiation of the

Caco-2 cells. Lastly, both COX-2 and Bcl-2 expression was downregulated in both the fish oil

and olive oil supplemented cells. The authors conclude by stating, “Fish oil and olive oil alter

different cellular processes leading to prevention of colorectal cancer development. COX-2 and

Bcl-2 could be partially responsible for some of the observed effects (Llor, 2003).” Notably, fish

oil not only prevented further cell proliferation of these cancer cells, but it also increased cellular

differentiation and apoptosis. These results help to explain some of the metabolic pathways that

are mediated by fish oil and shows us that fish oil effects gene expression of specific pro-

carcinogenic genes such as COX-2 and Bcl-2.

The final and most recent study supporting the use of omega-3 PUFAs to treat cancer

cachexia is a study by Murphy et al (Murphy, 2011). The purpose of Murphy’s study was to

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assess the effect of a fish oil intervention on body composition and weight compared to standard

of care during chemotherapy. There were forty patients in this study that were divided into two

groups. Sixteen patients were placed in the fish oil (2.2g of EPA/day) group and the remaining

24 were placed into the standard of care group. Body composition was measured using computed

tomography imaging. Blood and weight measurements were taken at baseline and throughout

chemotherapy.

The authors found that those in the standard of care group had an average weight loss of

2.3 kg, however those in the fish oil group maintained their weight. Secondly, the authors

reported that those who had the highest plasma EPA levels showed the greatest muscle gains.

There was a maintenance or gain in muscle mass in 69% of patients in the fish oil group. In

comparison, there was a maintenance of muscle mass in only 29% of patients in the standard of

care group (Murphy, 2011). Here we see that fish oil was superior to standard of care treatment

for maintaining body composition. Finally, the authors conclude that, “Nutritional intervention

with 2.2g of fish oil per day appears to provide a benefit over standard of care, resulting in

maintenance of weight and muscle mass during chemotherapy (Murphy, 2011).” This study

shows us that fish oil helps to maintain muscle mass in cancer patients, even during

chemotherapy treatment, very promising results for individuals with cancer cachexia.

Adding support to the argument for omega 3 PUFAs to treat cachexia is a double-blind,

placebo-controlled study looking that looked at advanced lung cancer patients (Finocchiaro,

2012). This study looked into how EPA and DHA would effect inflammation and oxidative

stress in lung cancer patients. They randomly divided 33 patients with non-small-cell lung cancer

into two groups. The first group received 850 mg of EPA/DHA and the second group received a

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placebo. The subjects were followed for 66 days and biochemical and anthropometric

measurements were taken at day 8, 22, and 66.

The results showed a significant reduction in inflammatory cytokines CRP, TNF-a, and

IL-6 in the n-3 group. Additionally, there was a significant reduction in plasma ROS compared

to the placebo group. Lastly, the study showed a slight increase in protein and energy intake in

the n-3 group and a significant increase in body weight in the treatment group. These positive

results support the use of n-3 PUFAs in cachexia treatment, however, the authors concluded that

there is still not enough clinical evidence to justify using n-3 PUFAs in a clinical setting.

Following this study was a study out of Taiwan that looked at omega-3 fatty acids, in

combination with micronutrients and probiotics, for their effects on weight gain in head and neck

cancer patients (Yeh, 2013). The authors examined the effectiveness of a nutritional supplement

for modifying body weight, albumin and prealbumin levels, and survival. After being randomly

assigned to receive either the nutritional supplement or an isocaloric control supplement, 68

subjects were followed for 3 months. Changes in body weight and albumin levels were

monitored.

The results indicated that the group taking the nutritional supplement enriched with n-3

fatty acids maintained their body weight throughout the treatment period whereas the control

group did not. Additionally, the treatment group had significantly improved albumin and

prealbumin levels compared to the control group. The ability of patients to maintain body weight

seen in this study supports an anti-cachectic effect of n-3 fatty acids. Moreover, a rise in protein

levels suggest another benefit of n-3 fatty acids and endorses their use in cachexia patients.

Lastly, a randomized control trial was published in 2014 that compared the use of an EPA

enriched nutritional supplement versus an isocaloric control to treat clinical and biochemical

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outcomes in patients with advanced non-small-cell lung cancer (Sanchez-Lara, 2014). This study

looked at the effects on body weight, inflammation, energy intake, quality of life, chemotherapy

response, and survival. The 96 patients were randomized and followed for 8 weeks, with

nutritional, clinical, and biochemical measurements taken after each round of chemo.

The omega-3 fatty acid supplemented group was able to maintain their weight and even

significantly increased their lean body mass, whereas the control group showed the opposite.

Inflammation was also improved in the treatment group, which displayed a significant reduction

in CRP and TNF-a levels, while the control group showed no changes. Energy and protein intake

was reduced in the control group but significantly increased in the treatment group. Additionally,

the treatment group showed reduced appetite loss and fatigue and an increased quality of life

compared to the control group. There were no significant differences in responses to

chemotherapy between groups.

In summary, the previous nine studies have created a very strong argument for the

effectiveness of omega-3 fatty acids found in fish oil to prevent cancer cachexia. We have seen

that fish oil has been effective in reversing cachexia in many different types of cancer including

pancreatic, breast, colon, and lung cancers. These studies have shown fish oil to inhibit weight

loss in a cachexia model, to delay the progression of cachexia in pancreatic cancer, and to restore

immunodeficiency and prolong survival of malnourished patients. Additionally, fish oil enriched

nutritional supplements caused pancreatic cancer patients to gain weight. Fish oil has also shown

to positively affect metabolic mediators of cachexia such as cytokine production, in addition to

being able to modulate gene expression of key mediators of cachexia such as COX-2 and Bcl-2.

Furthermore, these studies have shown that omega-3 PUFAs from fish oil increase the

effectiveness of chemotherapy such as doxorubicin without causing toxicity to the host cell, in

20

addition to fish oil providing a benefit over standard of care for preserving muscle mass and

improving body composition in patients with advanced cachexia. Most recently, randomized

control trials have showed us that omega-3 fatty acids are effective at reducing inflammation and

ROS levels, increasing total energy intake, and maintaining body weight, with the most recent

study showing an increase in lean body mass and an improvement in quality of life. When all of

these studies are taken into account we cannot deny that there is very strong evidence supporting

the use of omega-3 PUFAs from fish oil for the treatment of cancer cachexia. However, before

any conclusion can be made, the opposing side of this argument must be presented and all

relevant literature will be reviewed and discussed.

Review of Refuting Articles The earliest study that will be discussed was published in 1992 by Calder and

Newsholme (Calder, 1992). The aim of this study was to study the effects of fatty acids on

proliferation of blood lymphocytes and on IL-2 concentration. The authors wanted to look at the

effects on the immune system because numerous studies have shown fatty acids to be able to

modulate the immune system. Blood was drawn from five female subjects and transferred to

Leucoprep tubes for centrifugation. PUFAs were added to the tubes and after 48 hours the blood

was assayed for lymphocyte proliferation and IL-2 levels.

The results from the assays showed that EPA caused a significant inhibition of blood

lymphocyte proliferation. In addition, EPA also significantly decreased the concentration of IL-2

in blood lymphocytes. These findings are important to discuss because they cause a reduction in

the immune system after the addition of PUFAs. Lymphocyte proliferation has been shown to be

dependent on its ability to secrete IL-2. In this study PUFAs were shown to prevent the secretion

21

of IL-2, therefore halting lymphocyte proliferation and weakening the immune system. This

raises serious concerns for cancer patients who are already severely immunocompromised.

The following year another study was published that also looked at omega-3s effect on

the immune system, specifically IL-2 and mononuclear cells (Endres, 1993). This study by

Endres reinforced the previous study, finding similar results. Eighteen male volunteers were

given 18g (Each gram containing 153mg EPA and 103 mg DHA) of lipid concentrate per day

and continued this for six weeks. Blood was drawn at baseline and at the six-week mark and was

assessed for mononuclear cell proliferation and IL-2 production.

IL-2 production was remarkably reduced by 65% compared to baseline levels.

Furthermore, mononuclear cell proliferation mirrored the IL-2 response and showed a 70%

decrease compared to baseline. While these results may be useful in diseases that show an

overexpression of the immune system such as rheumatoid arthritis, these effects would be

detrimental to cancer patients who are already severely immunocompromised. The reduction in

immune function seen in these two early studies highlights one of the negative side effects of fish

oil supplementation.

The next study that will be reviewed showed that EPA had no effect on preventing

cachexia (Costelli, 1995). This study assessed if omega 3 polyunsaturated fatty acids, especially

EPA, effectively prevents hypercatabolism in tissues or changes tumor growth rates. Male wistar

rats were injected with Yoshida AH-130, a rapid growing hepatoma that causes weight loss and

tissue wasting. They were randomly divided into two groups, the first receiving daily EPA and

the second receiving a control diet. At 0, 4, and 7 days after injection of the hepatoma, the mice

were killed and tumor volume was assessed.

22

The hepatoma created a rapid loss of body weight and muscle in the rates. The rats in the

group receiving the EPA showed no alteration to the tumor growth rate compared to the control

group. Furthermore, the EPA was also ineffective at reducing the rate of wasting as compared to

the control group. The authors conclusion states, “The use of n-3 PUFA to prevent cancer

associated cachexia may be limited by the type of tumor. Very undifferentiated tumors are

probably insensitive to growth modulation by such agents (Costelli, 1995).” This study shows no

effect of EPA in reducing cachexia, at least in this tumor type, and weakens the argument for

using n-3 PUFAs to treat cachexia.

Moving ahead three years, we go to a study looking into the effects of PUFAs on colon

cancer in rats (Griffini, 1998). This study is one of the strongest advocates against the use of

omega 3 PUFAs in cancer patients. The purpose of this study was to investigate the effects of n-3

and PUFAs on the development of colon carcinoma metastasis in rat liver (Griffini, 1998). Wag-

Rij rats were divided into three dietary groups, one receiving a fish oil diet, one receiving a

safflower oil diet, and the last group receiving a low fat control diet. The rats were maintained on

their diets for three weeks. At the three-week mark, CC531 colon carcinoma cells were

transplanted into the rats. At 1 and 3 weeks, rats were euthanized and liver metastasis was

measured.

The data showed that at 1 week after transplantation there were 7x more metastasis in the

fish oil group compared to the low fat diet group. Additionally, at 3 weeks after transplantation

there were 10x more metastasis in the fish oil group compared to the low fat diet group. Adding

to these alarming results, the size of the metastasis in the fish oil group were 1000 fold larger

than those found in the low fat diet group. Griffini concludes, “N-3 PUFAs promote colon cancer

metastases in the rat liver. This finding has serious implications for the treatment of cancer

23

patients with fish oil to fight cachexia (Griffini, 1998).” This study is very alarming, showing

that n-3 PUFAs not only promoted extreme increases in number of metastases but also in the size

of the metastases.

In 2000 the Journal of Clinical Nutrition published a study out of the Netherlands that

investigated the effect of EPA on rates of lipolysis and lipid oxidation in cachectic patients and

healthy individuals (Zuijdgeest-Van Leeuwen, 2000). This was being investigated because it was

proposed that weight loss in cancer patients can be prevented through lipolysis inhibition. There

were 17 cancer patients and 16 healthy subjects, all randomized to receive either a EPA (6g/day)

or a placebo. The subjects were followed for seven days, with whole body lipolysis, palmitate

oxidation, and palmitic acid release being measured at baseline, day 2, and day 7 of the

intervention.

The authors reported no significant differences in lipolysis between groups. Secondly,

palmitate oxidation and palmitic acid release did not differ between the two groups. In

conclusion, the authors state, “Supplementation of EPA does not significantly inhibit lipolysis or

lipid oxidation in weight-losing cancer patients or in healthy subjects (Zuijdgeest-Van Leeuwen,

2000).” By showing fish oil is ineffective at reducing wasting of adipose tissue and preventing

lipolysis in cachectic patients, this study refutes the use of omega-3 PUFAs in treating cachexia.

The ensuing study in this review is just as bleak as the previous by Van Leeuwen. In

2003, Gut published a randomized double blind trial by Fearon et al. that assessed the effect of a

n-3 enriched supplement on weight loss (Fearon, 2003). This study compared a protein and

energy dense supplement enriched with n-3 fatty acids with an isocaloric control supplement.

They tested for effects on weight, lean body mass, dietary intake, and quality of life in cachectic

24

patients (Fearon, 2003). There were 200 patients in this trial, randomized to either receive the n-3

fatty acid or the control supplement and followed for eight weeks.

At the end of the 8-week intervention period there was no significant difference in the

two groups in terms of weight loss and lean body mass. Reduction in the rate of weight loss was

the same in both groups. Changes in lean body mass were also similar in both groups. The

authors concluding statement said, “Enrichment with n-3 fatty acids did not provide a therapeutic

advantage and both supplements were equally effective in arresting weight loss (Fearon, 2003).”

There was not a clear advantage and no difference in outcomes between the experimental and

control arms. We can see from this study that the use of omega-3 fatty acids to prevent cachexia

would be ill advised as it provides no additional benefit when compared to traditional treatments.

Jatoi lead a similar study in 2004 that investigated whether an EPA supplement would be

more effective than megestrol acetate at augmenting appetite and weight gain in patients with

cancer associated cachexia (Jatoi, 2004). This study randomly assigned 421 cachectic patients to

one of three groups. The first group received 1.09g EPA supplement plus a placebo, the second

group received 600 mg/d megestrol acetate plus an isocaloric supplement, and the third group

received both the EPA supplement as well as megestrol acetate. Weight measurements and

physical examinations were taken at baseline and weekly.

In terms of appetite improvement, there was no significant difference between the three

treatment arms. However, both of the treatment arms receiving megestrol acetate showed an

increase in functional assessment compared to the fish oil treatment arm. In addition, there was a

smaller percentage of patients who gained greater than 10% of baseline weight in the EPA group

compared to those in both megestrol acetate treatment groups. Lastly, there was no significant

differences in quality of life or survival between the two groups. In conclusion, “This EPA

25

supplement, either alone or in combination with megestrol acetate, does not improve weight or

appetite better than megestrol acetate alone (Jatoi, 2004).” EPA supplementation showed zero

improvements in any of the parameters measured compared to the other treatment arms. This

suggests no benefit in using omega-3 PUFAs to treat cancer cachexia.

Next up in the review of studies opposing the use of fish oil to treat cancer cachexia is a

randomized pilot study looking at advanced gastrointestinal cancer patients (Persson, 2005). The

objective of this pilot study was to investigate the effect of fish oil supplementation in

combination with melatonin and dietary advice on the biochemical variables known to develop

with cachexia, specifically cytokines. In addition, the authors examined clinical outcomes such

as body weight, quality of life, and food intake. The twenty-four patients were randomized to

receive either fish oil (4.9g DHA and 3.2g DHA), melatonin, or a combination. At baseline and

at 4 weeks plasma was analyzed for TNF-a, IL-6, IL-1B, IL-8, DHA, EPA, linoleic acid, and

arachidonic acid (Persson, 2005).

The results showed that there was no significant difference in any of the cytokines and no

difference in biochemical variables between the fish oil and melatonin groups. Moreover, there

was not a significant difference in energy intake, functional performance status, or survival

between the two groups. The authors conclude, “Fish oil, melatonin, and their combination, did

not produce substantial anti-inflammatory effects in cachectic patients with advanced

gastrointestinal cancer (Persson, 2005).” No benefits in inflammatory cytokine concentrations

were seen when fish oil was supplemented, contrary to what has been shown in previous fish oil

studies. These results oppose the use of fish oil for treating cancer cachexia, due to an absence of

an anti-inflammatory effect that they are commonly associated with.

26

The last study that will be covered in this review was published in 2006 in the Journal of

Clinical Oncology (Fearon, 2006). This article was double-blind, randomized, and placebo-

controlled. The purpose of this study was to compare EPA diester against a placebo for their

effects on lean body mass and weight in an eight-week intervention. This was a large study, with

518 patients being randomly assigned to one of three treatment arms to be followed for eight

weeks. The first group received 2g of EPA daily (n=175), the second group received 4g EPA

daily (n=172), and the final group received a placebo (n=171). Assessments were taken at 4 and

8 weeks.

The results indicated no significant changes in weight, survival, or nutritional variables.

Fearon concludes, “Overall, no statistically significant effects of treatment on the primary

endpoint of weight were observed…There were no other obvious differences in nutrition or

quality of life measures as a result of EPA diester administration...The results indicate no

significant benefit from single agent EPA in the treatment of cancer cachexia (Fearon, 2006).”

The current study, reporting no changes in body weight or quality of life by the addition of EPA,

illustrates that EPA is not useful for preventing cancer cachexia and provides a strong argument

against its use in this population.

Taking all of these studies into account we can see that there is also a strong argument

against the use of omega-3 PUFAs to treat cancer cachexia. Opposing the views of the previous

set of studies, these articles demonstrate that fish oil can potentially be quite harmful and

advocate that it should not be used to treat cachexia. The data from these original articles have

shown that fish oil can reduce the immune system by suppressing IL-2 production and

lymphocyte proliferation, has increased the number and size of colon metastases in an animal

model, is ineffective at preventing lipolysis, and is simply ineffective at modifying many clinical

27

outcomes such as quality of life, performance status, and survival. It is very important to

consider the results of these studies when assessing the use omega-3 fatty acids to treat patients

with cancer-induced cachexia.

DiscussionThis area of study has become a major controversy, especially in clinical practice. While

there are numerous studies showing that fish oil has positive effects on clinical outcomes of

cachexia, there are also just as many studies showing the opposite. For every study that came out

supporting the use of fish oil to treat cachexia, another article followed refuting its use. Because

of this dichotomy in the literature, oncologists have become torn on this issue, some saying that

yes, we should supplement fish oil in cachectic patients, whilst other clinicians are very opposed,

saying that it has no benefits and will only add to the financial burden of the patient. In addition

to the financial burden, some clinicians argue that it is already difficult to get cancer patients to

eat because of their loss of appetite and adding fish oil into their treatment plan will only

complicate this issue further. So what conclusions can be drawn after reviewing both sides of the

literature?

From reviewing both the supporting and opposing articles we can see that there is strong

evidence supporting both sides of this debate. In order to decide which side is more convincing

we must take a deeper look at each study and carefully examine the methodology and results and

compare strengths and weaknesses of each study. We can see that omega-3 fatty acids show very

positive results in the early cell and animal studies. However, when we look at the human studies

we can see that the results were a lot dimmer. Because the larger human trials show negative

results, I am persuaded to support the side against the use of omega-3 fatty acids. Cell and

28

animal studies are important for understanding the mechanisms, but phase 3 human studies are

much more applicable to a clinical setting. Cell and animal studies are easy to control for

confounding variables, but studying nutrition in humans can be very difficult because of our free

will. I will discuss both sides and why I believe that there currently is not enough evidence to

support the use of omega-3 fatty acids to treat cancer cachexia.

As discussed previously, almost all cell and animal models show that omega 3 PUFAs

produce a beneficial effect in a cachexia model. In cellular studies they were shown to reduce

inflammatory cytokine levels such as CRP, TNF-a, IL-1, and IL-6 and to reduce PIF activation

(Rondanelli, 2008). We have also seen that they can suppress the ubiquitin-proteasome pathway

and suppress the cancer cachetic factor (Jatoi, 2005). However, we also know that the immune

system can be compromised with the use of n-3 fatty acids. Lymphocyte proliferation was halted

in one study (Calder, 2002), and mononuclear cell production was greatly reduced in another

(Endres, 1993). Cancer patients typically have very weakened immune systems already so we

must carefully consider these studies when deciding whether or not to supplement n-3 fatty

acids. In terms of cell studies, the majority of the evidence supports the use of omega-3 fatty

acids for cachexia patients, but the possible reduction in immunity must be taken into account.

When we look at animal studies we see a similar pattern. The majority of evidence is in favor of

the use of omega-3 fatty acids to treat cachexia. However, one study did show 10x greater

number of colon metastasis in rats supplemented with fish oil compared to the control group

(Griffini, 1998). Cell and animal models, while important and useful, can only tell us so much.

The results of human studies have much more weight on the clinical applicability.

When we look at the earlier human studies we can see that there are many flaws. Many of

the studies had small number of subjects meaning the results can’t be generalized to the total

29

population group. The study by Wigmore only had 18 patients, the study by Gogos only had 15

subjects (Gogos, 1998), the study by Barber only had 18 subjects (Barber, 1999), and the study

by Barber in 2001 only had 20 patients (Barber, 2001). Although these sample sizes are

somewhat small we also have to keep in mind that there are many factors affecting participation

when dealing with advanced cancer patients. It is very difficult to get very sick patients to

volunteer for studies when they are undergoing chemotherapy and radiation, and this is also why

there is low compliance and a high dropout rate in some of these studies. While this is something

that can be very difficult to improve upon, it still remains a flaw of many of these studies

supporting the use of fish oil.

Furthermore, when we look at some of the larger clinical trials in humans we see that

they show no effect. A study using 91 patients compared fish oil to olive oil on its ability to

affect clinical outcomes in cachexia patients. In this well designed study there was absolutely no

change in appetite, energy intake, or functional status between the groups (Bruera, 2003).

Another large clinical trial (N=200) showed no difference in rate of weight loss between an

EPA-enriched supplement group and an isocaloric supplement group (Fearon, 2003). Lastly, a

large double blind study (N=421) showed that EPA had no added benefit for treating cachexia

compared to megestrol acetate alone (Jatoi, 2004). Each of these studies had much larger sample

sizes and better study designs than the earlier human studies. Some would argue that the dose of

fish oil was too small to see an effect and a weakness in these studies, but I do not believe that

this is the case. For this reason, these later trials hold more weight than the earlier trials showing

positive results. Based on this information, I am convinced that the use of omega-3 fatty acids is

ill advised for patients with cachexia.

30

The most recent randomized clinical trials began to show a trend in support of the use of

omega-3 fatty acids to treat cachexia. However, in one study, the intervention involved n-3

PUFAs, probiotics, and micronutrients (Yeh, 2013). It cannot be said for certain that the positive

results of this study were due to the omega-3 fatty acids. Another recent study showed that the

group receiving n-3 fatty acids showed a significant decrease in inflammatory cytokine and ROS

levels and an increase in energy intake (Finocchiaro, 2012). However, in their discussion the

authors state that they think the differences seen between the two groups are random and not

caused by the treatment. It is important to look closely at each study before drawing conclusions.

ConclusionIn summary, I believe that there is not enough evidence to support the use of omega-3

fatty acids for treating cancer-induced cachexia. While there are ample amounts of research

showing beneficial effects of omega-3 fatty acids, specifically in cell and animal models, the

larger, phase-3 clinical trials in humans show negative results and are more convincing.

Furthermore, after considering flaws in study design of the smaller human trials, I am more

swayed by the larger clinical trials that have better study designs and superior methodology.

Based on the reviewed evidence, omega-3 fatty acids may or may not have an effect depending

on numerous variables. Supplementing with fish oil, while being relatively safe and having very

minimal side effects, is simply not supported by the literature at this time. The answer is not a

definitive no at this point, but adding fish oil into a treatment regimen for an individual with

advanced cancer can possibly create more problems than it would solve and is ill advised. While

it can be highly appealing to some looking for anything to relieve the deleterious effects of

cachexia, we do not know enough about it yet and further research is necessary.

31

Future ResearchWhile there are currently hundreds of studies on this subject and our understanding of

certain aspects of it are plentiful, we must continue to develop future research in order to fully

understand this phenomenon before we can come to a definitive conclusion. The scientific

community has made leaps and strides in understanding how omega 3 fatty acids work over the

last 50 years, however, there are still some specific questions that we have yet to answer. In

addition to these questions, I believe that future studies should change the way certain parts of

the study methodology is carried out.

Future studies should address some of the following questions. How does the stage of

cachexia progression affect the effectiveness of omega 3 supplementation? For example, what

will the difference in outcome be if treatment is started in the pre-cachexia stage, vs. the cachexia

stage, vs. the refractory cachexia stage? Future studies also need to examine how other clinical

and nutritional variables play a role in how effective omega-3 fatty acids will be. How do

comorbidities, genotype, epigenetics, environment, and other dietary factors affect the outcome

of treatment? Furthermore, it would be beneficial to have a deeper understanding of the

underlying mechanisms omega 3s use to exert their effects in order to create more effective

treatment options. Lastly, there is simply a need for additional large scale, randomized clinical

trials in humans.

In terms of the methodology, all efforts in the future should be placed on making the

studies as equal as possible so that comparisons can be made. In the past, study designs are rarely

comparable and most studies are highly heterogeneous. Tolerability of the EPA/DHA source

should be made more tolerable so that there will be less dropout. Moreover, compliance with the

treatment needs to be better addressed in future studies to ensure the desired treatment effect is

32

taking place. Making these changes will ensure that more subjects are able to complete the trial

and the results are more legitimate.

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