Accepted Manuscript

TNF- and cancer cachexia: Molecular insights and clinical

implications

Hetal J. Patel, Bhoomika M. Patel

PII: S0024-3205(16)30685-3
DOI: doi: 10.1016/j.lfs.2016.11.033
Reference: LFS 15099
To appear in: Life Sciences
Received date: 24 September 2016
Revised date: 27 November 2016
Accepted date: 30 November 2016

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 ACCEPTED MANUSCRIPT

TNF- and Cancer Cachexia: Molecular insights and Clinical implications

Hetal J Patel,1 M. Pharm., Bhoomika M. Patel*2, Ph.D.
1
Apex Pharmacy, Arroyo Grand, California, USA
2
Institute of Pharmacy, Nirma University, India

* Corresponding author
Dr. Bhoomika M. Patel

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Assistant Professor
Institute of Pharmacy, Nirma University
Sarkhej-Gandhinagar Highway,

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Ahmedabad 382481, Gujarat, India
Phone No: + 91 2717 241900-04

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Fax No: + 91 2717 241916
Email: drbhoomikampatel@gmail.com NU
No. of figures: 04
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No. of Tables: 01
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Abstract
Cancer cachexia characterized by a chronic wasting syndrome, involves skeletal muscle loss
and adipose tissue loss and resistance to conventional nutritional support. Cachexia is
responsible for the reduction in quality and length of life of cancer patients. It also decreases
the muscle strength of the patients. The pro-inflammatory and pro-cachectic factors produced
by the tumor cells have important role in genesis of cachexia. A number of pro-inflammatory
cytokines, like interleukin-1 (IL-1), IL-6, tumor necrosis factor- alpha (TNF-) may have
important role in the pathological mechanisms of cachexia in cancer. Particularly, TNF- has

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a direct catabolic effect on skeletal muscle and causes wasting of muscle by the induction of
the ubiquitin-proteasome system (UPS). In cancer cachexia condition, there is alteration in

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carbohydrate, protein and fat metabolism. TNF- is responsible for the increase in

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gluconeogenesis, loss of adipose tissue and proteolysis, while causing decrease in protein,
lipid and glycogen synthesis. It has been associated with the formation of IL-1 and increases
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the uncoupling protein-2 (UCP2) and UCP3 expression in skeletal muscle in cachectic state.
The main aim of the present review is to evaluate and discuss the role of TNF- in different
metabolic alterations and muscle wasting in cancer cachexia.
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Key words: Cancer cachexia; tumor necrosis factor- alpha (TNF-); skeletal muscle wasting;
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acute phase response; metabolic abnormalities

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INTRODUCTION
Cancer cachexia, a metabolic syndrome is characterized by anorexia, loss of weight and
decreased muscle strength [1]. Decreased physical activity, poor quality of life, poor
performance status, increased risks of cancer treatment and high mortality rates are mainly
present in cancer cachectic patient which affects patients physiological and biochemical
balance [2]. Up to one half of all cancer patients may have cachexia and represents a
significant decreased physical activity and psychological burden [3]. Cachexia is associated

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with skeletal muscle protein loss and reduction of body lipid stores during metabolic process.
These metabolic changes in cachexia are to some extent facilitated by changes in
concentration of hormone present in the circulation including insulin, glucagon and

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glucocorticoids [4]. In addition, the tumor and host cells have competition for nutrients which

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leads to an enhanced catabolic state. Severe alterations in metabolic process are stimulated in
the host, including hyper metabolism leading to a reduced energy productivity [5].
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Various pro-inflammatory cytokines such as tumor necrosis factor - (TNF-), IL-6, IL-8,
interferon (IFN)- , parathyroid hormone- related peptides (PTHrP) and macrophage
migratory factor (MIF) also have important role in cancer cachexia [6]. TNF- is a cell
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signaling protein responsible for several metabolic derangements [7]. TNF- is released by
activated macrophages and by many other types of cell such as CD4+, neutrophils, mast cells,
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eosinophils and neurons. TNF- is able to induce apoptotic cell death, cachectic condition
and inflammatory response [8]. TNF- can promote most of the abnormalities found during
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cancer cachexia, i.e., loss of weight, loss of appetite, increased thermogenesis, alterations in
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carbohydrate, protein and lipid metabolism, insulin resistance and wasting of muscle by the
activation of breakdown of protein [9]. The present review aims to put forth the role of TNF-
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in cancer cachexia.
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CANCER CACHEXIA
Various pathophysiological changes involved in the progress of cancer cachexia are anorexia,
loss of body weight, metabolic alterations i.e. glucose, protein and lipid metabolism, systemic
inflammation, insulin resistance, oxidative stress and muscle protein degradation [10-12].
Several pro-inflammatory cytokines and pro-cachectic factors are considered as mediators of
the cachectic process [13]. Various cytokines responsible for the inflammation increase the
host systemic inflammatory response and pro-cachectic factors like proteolysis inducing
factor (PIF) and lipid mobilizing factor (LMF) have direct catabolic effect on host tissues
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during cancer cachexia [14]. Table 1 provides an overview of pathophysiological

mechanisms of cancer cachexia.

TNF- IN ANOREXIA AND BODY WEIGHT LOSS

Anorexia plays a vital role in accounting for malnourishment; perpetually has association
with cachexia during cancer. The cytokine involved for the progression of cancer cachexia is
TNF-. TNF- increases the corticotrophin-releasing hormone (CRH) level and decreases

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food intake (Figure 1). It also increases the firing of neurons, which, are sensitive to glucose
and, causes the reduction in food intake [15, 16].
Body weight loss is the noticeable clinical feature of cachexia in adults [17]. Body weight

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loss, which is more than 5- 10 % of original body weight is considered as a definition point

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for cachexia [18]. Generally body weight loss is responsible for nearly 30% deaths [19].
Different preclinical studies have reported that cytokines have ability to increase loss of body
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weight. It has been demonstrated that the nude mice showed anorexia, progressive wasting
and death when implanted with Chinese Hamster Ovary cells (CHO) in which human TNF
gene was used for the transfection in CHO cells [20]. It specifies that TNF- has important
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role in the progression of cachexia. Furthermore, the considerable amounts of TNF- has
been detected in the blood of rats with tumor [21]. In contrast, the experimental evidence by
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Mulligan et al. [19] reported that loss of body weight in animals possessing a murine
adenocarcinoma (MAC 16) is not associated with the cytokines TNF- or IL-6 [19]. A
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possible explanation for this could be that body weight loss occurs due to two major reasons:
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a) skeletal muscle wasting and b) adipose tissue loss and TNF- is reported to play a role in
both of these [22] (Figure 1).
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Skeletal Muscle Wasting

The decrease in the skeletal muscle mass is associated with the decrease in strength, energy
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and poor quality of life of patient [23, 24]. As muscle loss increases, there is a decreased
movement and individuality and also hospitalization rate increases [24]. The decreased
protein synthesis rates and increased protein degradation rates (protein turnover) is
responsible for the loss of skeletal muscle mass. TNF- activates this wasteful metabolic
process [25].
Several experimental evidences from preclinical studies suggest that TNF- has important
role in wasting of muscle during cancer cachexia [26, 27]. One experimental finding by Li et
al. [26] reported that there was decrease in total content of protein, including adult myosin
heavy chain fast-type (MHCf), in muscle by time and concentration depending manner,
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stimulated by TNF-. MHCf losses were not associated with change in rate of synthesis. This
observation suggest that TNF- stimulates degradation of myofibrillar proteins. Another
study has reported that acute treatment with recombinant TNF- in rats showed increased
degradation of protein and decrease synthesis of protein in soleus muscle (red), but not in
extensor digitorum longus (EDL) (white) muscle [28]. It has also been reported that there was
a decrease of body protein in diseased rats when they were treated with recombinant TNF-
for long time. Redistribution of protein and a marked depletion in content of muscle protein
occurs after long treatment with recombinant TNF- [29]. These findings suggest that TNF-

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decreases myofibrillar proteins mRNA levels and induces muscle wasting in later stage of
cancer cachexia. Furthermore, the engineered mice lacking in the TNF- receptor protein

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type I (TNFR1), when transplanted with Lewis lung carcinoma showed decreased muscle

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wasting compared with wild-type mice even with similar TNF- level in serum in both [30].
It is also suggested that TNFR1 subtype is involved in muscle protein degradation rather than
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TNFR2 and stimulate muscle wasting. Some experimental studies [31, 32] reported that
catabolism of muscle is stimulated by TNF- by activation of the ubiquitin proteasome
pathway. After an acute, intravenous injection of TNF-, both free and conjugated ubiquitin
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and mRNA levels of ubiquitin was increased in the intact rats limb muscles [31]. These
results are in agreements with the study of Llovera et al. [33], in which they have reported
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that TNF- showed increased in levels of ubiquitin mRNA in excised muscle in vitro [33].
TNF- promotes a complex array of post receptor signaling events which cause pleiotropic,
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cell-type-specific responses. The response of TNF- at cellular level is mediated by three

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major pathways. Out of three pathways, NF-kB induced catabolic signaling plays a key role
in protein degradation associated with cachexia. In this pathway, TNF- activates nuclear
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factor kappa B (NF-B) which is a primary mediator for the control of transcription and a
major applicant for signaling during catabolism [34]. TNF- rapidly promotes activation of
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NF-kB in cells of skeletal muscle, including differentiable myotubes [35, 36] and
undifferentiable myoblasts [37, 38]. TNF- binds to the type 1 TNF- sarcolemmal receptor
and triggers the events. This receptor is involved in the regulation of protein loss [39].
The activity of protein kinase C (redox-sensitive kinase) is stimulated by TNF- [35]. Rapid
conjugation of ubiquitin to muscle proteins is also caused by TNF- [36]. These events cause
the proteasomal degradation of I-kBa and translocate the stimulated NF-kB to the nucleus
after 15 min of exposure of TNF- [36]. NF-kB affects on the expression of genes which
regulate ubiquitin proteasome pathway (UPP) and undoubtedly promotes the loss of protein
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[40]. The activation of UPP is triggered by TNF- stimulated NF-kB signaling. It results into
the degradation of the greater part of proteins intracellularly and is responsible for regulated
proteolysis, progression of cycle of cell, regulation of transcription, and presentation of
antigen [34]. The proteins must first be directed for the conjugation to many ubiquitin
molecules before they can be degraded by the UPP. For the conjugation, ubiquitin-activating
enzyme (E1) first activates ubiquitin and is then shifted to the ubiquitin carrier protein (E2)
active site. Ubiquitin conjugating enzymes (E3 or E3 protein ligase) is recognized by the

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bound E2, which allows the occurrence of conjugation reactions by the formation of
ubiquitins chain by attaching with each other and the protein substrate. The proteasome can
recognize a selected protein only after it is targeted by ubiquitin and then processed into

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smaller peptides [41, 42]. During proteolysis, there are three E3 protein ligases which are

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active in atrophy of muscle, namely, E3, MAFbx/atrogin-1 and MuRF1 [43- 47] (Figure 2a).
This pathway has been stimulated both in preclinical models and cancer cachectic patients
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[48- 51]. There is increase in components of the UPP in pre-cachectic patients. It suggests
that the system contributes to atrophy of muscle in patients with cachexia and also have a
vital role in the progression of the disease [48, 52]. There is increase in the activity of the
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UPP in catabolic states by increased expression of ubiquitin protein ligases [53].

Overall, these data indicate that TNF- can promote degradation of protein by directly acting
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on mature muscle. The ROS and NF-B are the early mediators of TNF- action. Both these
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components are responsible for the inflammatory response, characterized by the components
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of the degradation pathway for protein and add catabolic cytokine input to skeletal muscle.
Since, many cellular responses are mediated by ROS and NF-B, identification of more
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muscle specific downstream targets are necessary for the cachexia treatment. Further research
is required to establish targets which are muscle-specific.
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METABOLIC ALTERATIONS AND TNF-

In cachexia condition, there is metabolic alteration, which, causes the eventual increase in
expenditure of energy, systemic stress and interference of normal function of cells. The
presence and severity of cachexia correlates poorly with size of tumor. The factors produced
by the tumor or by the body in response to the tumor result in alteration in metabolism [54].
Altered lipid and carbohydrate metabolism and insulin resistance are key factors of metabolic
alterations of cancer cachexia in which TNF- plays an important role [55]. Additionally,
along with other cytokines, TNF- modulates nitrogen metabolism in cachexia [56].
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Altered lipid metabolism

In cancer bearing patients, there is an elevated level of circulating lipid. The increased level
of both triacylglycerol and cholesterol lead to hyperlipedemia in cancer-bearing states. The
decreased activity of lipoprotein lipase (LPL) causes the hypertriglyceridemia which causes
reduction in plasma clearance of both endogenous and exogenous triacylglycerols [57]. An
important link between decrease in activity of LPL and hypertriglyceridemia is present in
animals having tumor and responsible for with a high degree of cachexia [58]. Along with

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these metabolic processes, transport of glucose and de novo lipogenesis are inhibited in the
tumor tissue. Results from the studies indicate that loss of adipose tissue occurs due to
lipolysis or fat atrophy which is characterized by changes in metabolism of lipid [59- 61].

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Metabolic changes in the lipid result in excessive decrease of total fat mass, higher rate of

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lipolysis, total oxidation of fatty acids and higher levels of lipid, triglyceride and cholesterol
in blood. These are mediated by decrease in activity of LPL and increase in the activity of
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hormone-sensitive lipase (HSL) [62]. Atrophy of fat is due to the lipid mobilization that
constitute 95% of volume of fat cell. Specifically, free fatty acids (FFAs) and glycerol are
released in the circulation after the hydrolysis of triglycerides [63]. This reaction is catalyzed
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by the rate limiting enzymes HSL and adipose triglyceride lipase (ATGL). In cancer cachexia
patients, there is increase in both HSL activity and plasma FFA and glycerol [64]. The cyclic
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AMP (cAMP) is produced by the enzyme adenylate cyclase, which, further stimulates protein
kinase A (PKA), which in turn phosphorylates and produces activation of HSL (Figure 2b).
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TNF- is a potent inhibitor of lipogenesis. It suppresses the synthesis of transcription factor

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essential for the adipocyte differentiation [65]. Several in vitro and in vivo studies have
reported the involvement of TNF- in lipolysis by decreasing LPL activities. The activity of
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LPL was decreased by TNF- in 3T3-L1 cells [66]. Another study reported that the decrease
in mRNA level of LPL is caused by TNF- [67]. Fried and Zechner [68] demonstrated that
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the suppression of LPL activity was produced by TNF- in a dose-dependent manner in

human adipose tissue of human and organ culture was used to maintain adipose tissue.
Similarly Kawakami et al. [69] demonstrated that lipolysis was increased by the addition of
TNF- to 3T3-L1 cells alongwith a decrease in LPL activity. Ruan [70] have shown that the
cultivation of 3T3-L1 cells in the presence of TNF- showed an alterations in expression of
gene that are necessary for sensitivity of insulin and adipogenesis. Furthermore, TNF-
suppressed the expression of gene responsible for various nuclear transcription factors that
are required for differentiation and function of adipocyte, including Peroxisome proliferator-
activated receptor (PPAR), retinoid X receptor alpha (RXR) and CCAAT enhancer
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binding protein alpha (C/EBP) [65]. These findings indicate the major role of TNF- in
adipose tissue loss and in the development of cancer cachexia. TNF- and IL-1 are also
responsible for the inhibition of transport of glucose in adipocytes and subsequently reduce
the substrates availability for lipogenesis [71].
One study reported that there was decrease in LPL activity of adipose tissue when TNF- was
administered in rat, mouse and guinea pig in vivo [67, 72]. Due to decreased activity of LPL,
there was reduced adipose tissue uptake of exogenous lipid and increased triacylglycerols

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circulating level in the rat. This results due to the stimulation of adipose tissue lipolysis
following increased release of very-low density lipoproteins (VLDL) from the liver [73]. The
experimental study by Machado et al. [74] has shown that there was a significant increase in

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macrophage infiltration in white adipose tissue in rats when transplanted with the Walker 256

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carcinosarcoma and observation was made on the seventh day after the tumor inoculation.
Moreover, more TNF- was secreted by these cells when cultivated with serum from a
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Walker 256 tumor transplanted animal. TNF- is also reported to activate mitogen activated
protein kinase and extracellular signal related kinase and thereby stimulate lipolysis [75].
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Carbohydrate metabolism
Most solid tumors have hypoxic condition in certain region. Glucose metabolism under the
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hypoxic conditions produce lactate in large amounts, which, in the liver, gets converted back
into glucosethe Cori cycle. The lactate levels in tumor have been shown to be responsible
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for the metastasis, recurrence of tumor and decreased survival of patient (Figure 3). The
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cancer patients have marked metabolic alterations in carbohydrate in non-tumor tissues, in

particular the liver. It is because the glucose is used as the primary source of energy by tumor
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tissues [76]. The activity of cori-cycle is increased by 20%. In cachectic cancer patients,
glucose turnover is 50% and it accounts for the disposal of 60% of the lactate produced by
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the tumor [77]. Six ATP molecules are used for the gluconeogenesis from lactate for every
glucose-lactate cycle and is very energy incompetent for the host. In patients with lung and
pancreatic cancer, the energy expenditure is increased due to the futile cycle [78, 79]. In
weight-losing cancer patients, there was a 40% increase in production of glucose in liver
(hepatic gluconeogenesis) [81].
The utilization of glucose and formation of lactate are stimulated by TNF- by activation of
the substrate cycle between the enzymes phosphofructokinase and fructose-1, 6-
bisphosphatase, which therefore increases utilization of glucose and glycogen with a
consequent rise in lactate formation [82]. One study has shown that there was a rapid increase
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in production of lactate and in metabolism of glucose by TNF- in L6 myocytes in culture. It

has also been shown that action of TNF- in cultured myocytes is linked with a futile cycle
activation [81]. Other substance like amino acids resulting from myofibrillar proteins break
down in skeletal muscle, also cause the rise in gluconeogenesis in patients with cancer. TNF-
and IL-1 are responsible cytokines for the myofibrillar proteins breakdown in skeletal
muscles [82].
Insulin Resistance

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Insulin resistance has been considered as an important mechanism for the muscle wasting in
cancer cachexia. Insulin resistance is associated with the rise in degradation of protein and
skeletal muscle wasting [83]. In cancer cachexia, degradation of muscle protein is affected by

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a number of pathways, but evidence suggests that the UPP is particularly important,

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involving the support of caspase-3 [84]. Importantly, a number of signaling molecules are
activated by the insulin signaling pathway that overlap with the UPP. In insulin resistant
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states, there is decrease in phosphatidylinositol 3- kinase (PI3K) activity, which leads to
decreased Akt phosphorylation. Lower levels of pAkt is associated with increase in atrogin-1
and MuRF-1 mRNA expression, two important components of the UPP. Through this
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pathway, insulin resistance can cause muscle protein degradation [85].

Induction of TNF- mRNA and down regulation of mRNA of glucose transporter 4 (GLUT4)
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can result in insulin resistance in patients with cancer [86]. TNF- is responsible for
decreased insulin sensitivity and contributes to insulin resistance in cancer. The intracellular
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and molecular mechanisms like stimulation of stress-related protein kinases (JNK) and
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inhibitor kappa beta kinase beta (IKK)/NF-B pathway are responsible for the insulin
resistance induced by TNF- [87]. The increased levels of TNF- in circulation stimulate cell
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surface TNF- receptors leading to activation of JNK. It shows a vital role in insulin
resistance. JNK activation cause the phosphorylation of insulin receptor substrate (IRS)-1 at
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serine (Ser) sites and may impair insulin action [88- 90]. Signaling of insulin receptor that
generally occurs by cascade of a tyrosine kinase is repressed by phosphorylation of counter-
regulatory serine/threonine. Translocation of NF-B is caused by the IKK activation, which
results in a feed-forward loop that increases the synthesis of inflammatory mediators
including TNF- that is responsible for the induction of insulin resistance [91]. Insulin
resistance increases the proteolysis activity by increasing the expression of UPP components.
It can result in wasting of skeletal muscle in cancer cachectic patients [83]. The experimental
study by Stephens et al [92] reported that there was significant reduction ( 80%) in IRS-1
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and GLUT-4 mRNA after prolong exposure of TNF- on 3T3-L1 adipocytes, indicating
resistance to insulin is caused by TNF- in adipocytes.
Some clinical studies have also reported that the patients with different types of tumors
showed presence of insulin resistance due to impaired glucose tolerance and decreased
sensitivity of insulin. The involvement of TNF- in induction of insulin resistance was
confirmed by Noguchi et al. [86]; they suggested that increase in mRNA of TNF- and down
regulation of mRNA of GLUT4 can result in insulin resistance in peripheral tissues of cancer

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patients [86]. Furthermore, another study has reported that the concentration of TNF-alpha in
serum and insulin resistance (peripherally) have positive correlation when studied in 11
gastrointestinal cancer patients [93].

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Therefore, since insulin resistance is considered as an early sign of the cancer cachexia

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development, the treatment of this metabolic alterations by targeting TNF- may be able to
prevent the progress of cachexia.
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SYSTEMIC INFLAMMATION
Systemic inflammation is another important mechanism of cachexia. In systemic
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inflammation, there is production of C-reactive protein (CRP) and fibrinogen. These are
known as acute-phase proteins (APP). CRP binds to the exposed ligand present on affected
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cells and has been associated in wasting of skeletal muscle [94].

TNF- is considered as the major mediator of APP because TNF- is involved in the muscle
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protein breakdown and large secretion of amino acids mainly alanine and glutamine from
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skeletal muscle. The amino acids release is also stimulated by an inhibition of transport of
amino acid into skeletal muscle [58]. Alanine is used for the gluconeogenesis and APP
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synthesis in the liver, while glutamine is used by the tumor to maintain both its energy and
nitrogen demand. Increased production of APP in liver is partly responsible for the protein
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catabolism in the skeletal muscle [95]. In this way, TNF- contributes for the production of
APP (figure 4).
The APR is responsible for the inflammation present during cachexia [96, 97] and the
decreased quality of life and increased mortality in patients [98-100]. Patients having
pancreatic, lung or esophagus cancer, may be associated with an APP response [101]. An
increased APR was present in approximately 50% of patients having solid epithelial cancers
[98]. This APR has been associated with hypermetabolism, increased expenditure of resting
energy and decreased energy intake in patients with pancreatic cancer [101].
CONCLUSIONS
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Cancer cachexia is a multifactorial condition that involves the skeletal muscle loss and
adipose tissue loss, a dysregulated metabolic state and increased basal energy expenditure. It
correlates with poor quality of life, poor performance status and high mortality rate in cancer
patients. Although much research have been done for treatment of cachexia, the gap still
exists in management of cachexia. With the advancement in molecular biologic techniques,
the role of TNF- in cancer cachexia has been studied. TNF- plays an important role in all
key mechanisms viz. skeletal muscle loss, adipose tissue loss, alterations in carbohydrate,

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protein and lipid metabolism, insulin resistance, systemic inflammation in cancer cachexia.
Moreover, entire pathway of each mechanism with specific action of TNF- has been
established. Hence, future research should be directed towards altering the associated

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pathway and modifying the response of TNF- so as to achieve the therapeutic approach for

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treatment of cancer cachexia. On long term, such research may result in establishment of
therapeutic management of cachexia which may improve morbidity and mortality.
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ACKNOWLEDGEMENT: We are thankful to Science Engineering and Research Board
(SERB), Government of India for providing financial assistance (Ref No.: SB/YS/LS-
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144/2014).
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CONFLICT OF INTEREST
The authors declare no conflict of interest and financial disclosures.
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FIGURE LEGENDS
Figure 1: Role of TNF- in anorexia and body weight loss in cancer cachexia. TNF-- tumor
necrosis factor-alpha; CRH- corticotrophin releasing hormone; LPL- lipoprotein lipase
Figure 2: TNF- mediated (a) activation of ubiquitination and protein degradation resulting into
skeletal muscle wasting. TNFR1- TNF- receptor protein type 1; PKC- protein kinase C; NF-
B- nuclear factor kappa B; IKK- inhibitor kappa beta kinase beta; MAFbx- muscle specific
F- box; MURF1- muscle specific ring finger 1 (b) lipolysis leading adipose tissue loss. TG-

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triacylglycerol; HSL- hormone sensitive lipase; PKA- protein kinase A; FFA- free fatty acid
Figure 3: Role of TNF- in carbohydrate metabolism.

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Figure 4: TNF- induced acute phase protein (APP) response.

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Table 1: Various pathophysiological mechanisms of cancer cachexia.

Sr. Pathophysiological mechanisms of cancer cachexia

No.
Key mechanisms Resultant effects
1. Anorexia Malnutrition
2. Body weight loss Skeletal muscle loss

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Adipose tissue loss
3. Metabolic abnormalities Gluconeogenesis, proteolysis,

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(alteration in carbohydrate, lipolysis
protein and lipid metabolism )

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4. Insulin resistance Muscle protein degradation and
muscle wasting
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5. Systemic inflammation Acute phase protein response
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Graphical abstract

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