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NF-κBp65 and Expression of Its Pro-Inflammatory Target Genes Are Upregulated in the Subcutaneous Adipose Tissue of Cachectic Cancer Patients.

Camargo RG, Riccardi DM, Ribeiro HQ, Carnevali LC, de Matos-Neto EM, Enjiu L, Neves RX, Lima JD, Figuerêdo RG, de Alcântara PS, Maximiano L, Otoch J, Batista M, Püschel G, Seelaender M - Nutrients (2015)

Bottom Line: It secretes several cytokines capable of directly regulating intermediate metabolism.A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB).Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Group, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 1524-Cidade Universitária, Sao Paulo, 05508-000, Brazil. rodolfogcamargo@usp.br.

ABSTRACT
Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB). We have examined the gene expression of the subunits NF-κBp65 and NF-κBp50, as well as NF-κBp65 and NF-κBp50 binding, the gene expression of pro-inflammatory mediators under NF-κB control (IL-1β, IL-6, INF-γ, TNF-α, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-κBp65 and its target genes expression (TNF-α, IL-1β, MCP-1 and IκB-α) were significantly higher in cachectic cancer patients. Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-κB pathway plays a role in the promotion of WAT inflammation during cachexia.

No MeSH data available.


Related in: MedlinePlus

Western blot for nuclear and cytoplasmic protein extraction purity confirmation: β-Tubulin—51kd (A) and Lamin A—69 kd (B).
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nutrients-07-04465-f005: Western blot for nuclear and cytoplasmic protein extraction purity confirmation: β-Tubulin—51kd (A) and Lamin A—69 kd (B).

Mentions: Systemic inflammation is a central feature of cancer cachexia [16,20,21,22]. Circulating pro-inflammatory mediators such as Il-6, TNF-α and acute phase proteins (CRP) are upregulated in cachectic patients and correlate positively with weight loss and poor prognosis. In this study, CRP concentration was significantly higher in cachectic cancer patients. This acute-phase protein has been described as a marker of systemic inflammation and is also considered as part of cachexia diagnostic criteria [16]. Our results reinforce the importance of circulatory pro-inflammatory mediators as cachectic markers and corroborate previous studies with cancer patients [23]. The search for non-invasive cachexia markers is mandatory, and would warrant earlier intervention, thus preventing the onset of the symptoms and adverse prognosis. In this study, quality of life was evaluated by the application of the QLQ-C30 questionnaire. Patients reported diminished quality of life, which compromises the treatment adherence and survival. The three clusters analyzed in this questionnaire: Functionality (physical, cognitive, emotional and social), Symptomatic (fatigue, pain, nausea and vomiting) and Global health were all impaired in the cachectic cancer patients. Amongst the symptomatic cluster, fatigue is the main declared symptom and is usually present in more than 75% of cachectic patients [24]. It is accepted that systemic inflammation markedly contributes to the worsening of cachexia prognosis. Several peripheral organs suffer the consequences of inflammation triggered by the high circulatory level of pro-inflammatory mediators, that, in turn, induce in peripheral and central organs the activation of the inflammatory signaling pathways, such as the NF-κB pathway [25]. The NF-κB/Rel family of proteins, described first by Sen and Baltimore in 1986 [26] consists of transcription factors intensely studied due to the major implication as key mediators of a wide variety of cellular responses associated mainly with inflammation, infection and apoptosis. These include the stimulation of the expression of pro-inflammatory mediators such as TNF-α, IL-6, INF-δ, IL-1β, of chemokines such as MCP-1, and of reactive oxygen species (ROS) [12,13,27]. The relationship of the NF-κB signaling pathway with poor prognosis of cancer cachexia has already been extensively studied in the muscle, where increased NF-κB signaling has been described in patients [28]. Similarly, in patients under treatment for lung cancer, circulating pro-inflammatory mediators were associated with the activation of the NF-κB signaling pathway in the muscle [29]. Moreover, the importance of this transcription factor was confirmed by pharmacological inhibition, which was demonstrated to be an effective tool to reduce muscle proteolysis and consequently, atrophy [30,31]. Besides the muscle, other organs such as the liver, the brain, the gut and the adipose tissue are affected by cachexia. Adipose tissue metabolism is impaired and extensive lipolysis is observed [32]. WAT actively contributes to the inflammatory state in cachexia by actively secreting pro-inflammatory mediators [4,6,33]. Despite being recognized as an active player in cachexia, no information is available in the literature about the role of NF-κB in the development and maintenance of local inflammation of the adipose tissue. In the present study we demonstrate for the first time, that gene expression of NF-κBp65, which is a subunit of the one of the most important transcription factors that induce pro-inflammatory mediator gene expression, is upregulated in the subcutaneous adipose tissue of cachectic cancer patients, compared with controls. This is a strong indication of the role of NF-κBp65 in the promotion of inflammation in the subcutaneous adipose tissue in cachectic cancer patients. To confirm such participation of NF-κBp65 in the regulation of the adipose tissue inflammation, an assay was performed to evaluate NF-κBp65 and NF-κBp50 binding capacity to its promoter region of the DNA. This assay may be envisaged as a ‘snapshot’ of the cellular nucleus subunits NF-κBp65 and NF-κBp50 capacity of action, at the moment of tissue collection. Previous experiments of our group showed that the NF-κBp65 binding to its promoter region is higher, but not statistically significant among groups, while NF-κBp50 did not differ among groups (Figure A1). This assay, nevertheless, does not provide optimal evidence of total binding rate, as NF-κB dimer activity presents a fast up-regulation in the nucleus and then decays rapidly, migrating back to the cytoplasm, where it is again sequestered by its inhibitory protein, IκB-α. This is described as a rapid and dose-dependent response, that involves phosphorylation and subsequently proteasome degradation of the NF-κB inhibitory protein, IκB-α, which leaves the transactivation domain of the NF-κB dimer free to translocate to the nucleus and exert its functions as a transcription factor [34]. This rapid and transient stimulus, however, is sufficient to alter gene expression and cause prolonged changes in the NF-κB target proteins mRNA levels [35]. This led us to hypothesize that actually the best experiment to examine NF-κBp65 activity would rather consist of sequential evaluation of binding at different collection times; what is, unfortunately not possible in a human study due to ethical limitations. An alternative, however, would be the analysis of the expression of its target genes. Thus, we proceeded with the gene expression analysis of the NF-κBp65 pro-inflammatory target genes by qPCR, having found that IL-1β, TNF-α and MCP-1 expression were upregulated in cachectic cancer patients compared with controls. This is a strong indication that the local subcutaneous adipose tissue inflammation described in cachectic cancer patients may be mediated by the increased expression and activity of NF-κBp65. Considering that several inflammatory signaling pathways work in concert in promoting inflammation and, in order to verify the specific relationship between NF-κBp65 and the induction of pro-inflammatory genes expression, we performed Spearman’s correlation tests. The results show that all genes described as NF-κBp65 targets present a positive correlation with the expression of NF-κBp65 in the patients’ subcutaneous adipose tissue, including its inhibitor IκB-α, which is significantly more expressed in cachectic cancer patients, as compared with controls (Figure 4). These data strongly corroborate the NF-κB target gene expression results obtained in the study. Furthermore, NF-κBp65 is the most active protein able to induce IκB-α expression [36] and the accumulation of newly synthesized IκB-α is described as a pivotal factor in the termination of NF-κB activity and shuttling NF-κB complexes from the nucleus back to the cytoplasm [36]. Therefore, parallelism between NF-κBp65 and IκB-α, in a feedback mechanism was expected. This result was a confirmation of previous studies that pointed out a role of the NF-κB signaling pathway in the promotion of an inflammatory state related with cachexia. Despite these very encouraging results, other signaling pathways such as the signal transducer and activator of transcription 3 (STAT3), the c-Jun N-terminal kinase (JNK), p38 and AP-1 are also of interest in WAT inflammation. While in this study we focused solely on the NF-κB signaling pathway, these other pro-inflammatory pathways may not be disregarded, as they are known to participate actively as co-players of the worsened prognosis in cachexia [37,38,39]. The limitations of the study should be acknowledged. There was no measurement of the patient’s lean body mass, although the patients and groups presented similar BMI. Owing to human tissue sample implicit variation, some of the analyses were not performed with the total number of patients formerly enrolled as some samples fell out of the detection range of assays.


NF-κBp65 and Expression of Its Pro-Inflammatory Target Genes Are Upregulated in the Subcutaneous Adipose Tissue of Cachectic Cancer Patients.

Camargo RG, Riccardi DM, Ribeiro HQ, Carnevali LC, de Matos-Neto EM, Enjiu L, Neves RX, Lima JD, Figuerêdo RG, de Alcântara PS, Maximiano L, Otoch J, Batista M, Püschel G, Seelaender M - Nutrients (2015)

Western blot for nuclear and cytoplasmic protein extraction purity confirmation: β-Tubulin—51kd (A) and Lamin A—69 kd (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488796&req=5

nutrients-07-04465-f005: Western blot for nuclear and cytoplasmic protein extraction purity confirmation: β-Tubulin—51kd (A) and Lamin A—69 kd (B).
Mentions: Systemic inflammation is a central feature of cancer cachexia [16,20,21,22]. Circulating pro-inflammatory mediators such as Il-6, TNF-α and acute phase proteins (CRP) are upregulated in cachectic patients and correlate positively with weight loss and poor prognosis. In this study, CRP concentration was significantly higher in cachectic cancer patients. This acute-phase protein has been described as a marker of systemic inflammation and is also considered as part of cachexia diagnostic criteria [16]. Our results reinforce the importance of circulatory pro-inflammatory mediators as cachectic markers and corroborate previous studies with cancer patients [23]. The search for non-invasive cachexia markers is mandatory, and would warrant earlier intervention, thus preventing the onset of the symptoms and adverse prognosis. In this study, quality of life was evaluated by the application of the QLQ-C30 questionnaire. Patients reported diminished quality of life, which compromises the treatment adherence and survival. The three clusters analyzed in this questionnaire: Functionality (physical, cognitive, emotional and social), Symptomatic (fatigue, pain, nausea and vomiting) and Global health were all impaired in the cachectic cancer patients. Amongst the symptomatic cluster, fatigue is the main declared symptom and is usually present in more than 75% of cachectic patients [24]. It is accepted that systemic inflammation markedly contributes to the worsening of cachexia prognosis. Several peripheral organs suffer the consequences of inflammation triggered by the high circulatory level of pro-inflammatory mediators, that, in turn, induce in peripheral and central organs the activation of the inflammatory signaling pathways, such as the NF-κB pathway [25]. The NF-κB/Rel family of proteins, described first by Sen and Baltimore in 1986 [26] consists of transcription factors intensely studied due to the major implication as key mediators of a wide variety of cellular responses associated mainly with inflammation, infection and apoptosis. These include the stimulation of the expression of pro-inflammatory mediators such as TNF-α, IL-6, INF-δ, IL-1β, of chemokines such as MCP-1, and of reactive oxygen species (ROS) [12,13,27]. The relationship of the NF-κB signaling pathway with poor prognosis of cancer cachexia has already been extensively studied in the muscle, where increased NF-κB signaling has been described in patients [28]. Similarly, in patients under treatment for lung cancer, circulating pro-inflammatory mediators were associated with the activation of the NF-κB signaling pathway in the muscle [29]. Moreover, the importance of this transcription factor was confirmed by pharmacological inhibition, which was demonstrated to be an effective tool to reduce muscle proteolysis and consequently, atrophy [30,31]. Besides the muscle, other organs such as the liver, the brain, the gut and the adipose tissue are affected by cachexia. Adipose tissue metabolism is impaired and extensive lipolysis is observed [32]. WAT actively contributes to the inflammatory state in cachexia by actively secreting pro-inflammatory mediators [4,6,33]. Despite being recognized as an active player in cachexia, no information is available in the literature about the role of NF-κB in the development and maintenance of local inflammation of the adipose tissue. In the present study we demonstrate for the first time, that gene expression of NF-κBp65, which is a subunit of the one of the most important transcription factors that induce pro-inflammatory mediator gene expression, is upregulated in the subcutaneous adipose tissue of cachectic cancer patients, compared with controls. This is a strong indication of the role of NF-κBp65 in the promotion of inflammation in the subcutaneous adipose tissue in cachectic cancer patients. To confirm such participation of NF-κBp65 in the regulation of the adipose tissue inflammation, an assay was performed to evaluate NF-κBp65 and NF-κBp50 binding capacity to its promoter region of the DNA. This assay may be envisaged as a ‘snapshot’ of the cellular nucleus subunits NF-κBp65 and NF-κBp50 capacity of action, at the moment of tissue collection. Previous experiments of our group showed that the NF-κBp65 binding to its promoter region is higher, but not statistically significant among groups, while NF-κBp50 did not differ among groups (Figure A1). This assay, nevertheless, does not provide optimal evidence of total binding rate, as NF-κB dimer activity presents a fast up-regulation in the nucleus and then decays rapidly, migrating back to the cytoplasm, where it is again sequestered by its inhibitory protein, IκB-α. This is described as a rapid and dose-dependent response, that involves phosphorylation and subsequently proteasome degradation of the NF-κB inhibitory protein, IκB-α, which leaves the transactivation domain of the NF-κB dimer free to translocate to the nucleus and exert its functions as a transcription factor [34]. This rapid and transient stimulus, however, is sufficient to alter gene expression and cause prolonged changes in the NF-κB target proteins mRNA levels [35]. This led us to hypothesize that actually the best experiment to examine NF-κBp65 activity would rather consist of sequential evaluation of binding at different collection times; what is, unfortunately not possible in a human study due to ethical limitations. An alternative, however, would be the analysis of the expression of its target genes. Thus, we proceeded with the gene expression analysis of the NF-κBp65 pro-inflammatory target genes by qPCR, having found that IL-1β, TNF-α and MCP-1 expression were upregulated in cachectic cancer patients compared with controls. This is a strong indication that the local subcutaneous adipose tissue inflammation described in cachectic cancer patients may be mediated by the increased expression and activity of NF-κBp65. Considering that several inflammatory signaling pathways work in concert in promoting inflammation and, in order to verify the specific relationship between NF-κBp65 and the induction of pro-inflammatory genes expression, we performed Spearman’s correlation tests. The results show that all genes described as NF-κBp65 targets present a positive correlation with the expression of NF-κBp65 in the patients’ subcutaneous adipose tissue, including its inhibitor IκB-α, which is significantly more expressed in cachectic cancer patients, as compared with controls (Figure 4). These data strongly corroborate the NF-κB target gene expression results obtained in the study. Furthermore, NF-κBp65 is the most active protein able to induce IκB-α expression [36] and the accumulation of newly synthesized IκB-α is described as a pivotal factor in the termination of NF-κB activity and shuttling NF-κB complexes from the nucleus back to the cytoplasm [36]. Therefore, parallelism between NF-κBp65 and IκB-α, in a feedback mechanism was expected. This result was a confirmation of previous studies that pointed out a role of the NF-κB signaling pathway in the promotion of an inflammatory state related with cachexia. Despite these very encouraging results, other signaling pathways such as the signal transducer and activator of transcription 3 (STAT3), the c-Jun N-terminal kinase (JNK), p38 and AP-1 are also of interest in WAT inflammation. While in this study we focused solely on the NF-κB signaling pathway, these other pro-inflammatory pathways may not be disregarded, as they are known to participate actively as co-players of the worsened prognosis in cachexia [37,38,39]. The limitations of the study should be acknowledged. There was no measurement of the patient’s lean body mass, although the patients and groups presented similar BMI. Owing to human tissue sample implicit variation, some of the analyses were not performed with the total number of patients formerly enrolled as some samples fell out of the detection range of assays.

Bottom Line: It secretes several cytokines capable of directly regulating intermediate metabolism.A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB).Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Group, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 1524-Cidade Universitária, Sao Paulo, 05508-000, Brazil. rodolfogcamargo@usp.br.

ABSTRACT
Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB). We have examined the gene expression of the subunits NF-κBp65 and NF-κBp50, as well as NF-κBp65 and NF-κBp50 binding, the gene expression of pro-inflammatory mediators under NF-κB control (IL-1β, IL-6, INF-γ, TNF-α, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-κBp65 and its target genes expression (TNF-α, IL-1β, MCP-1 and IκB-α) were significantly higher in cachectic cancer patients. Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-κB pathway plays a role in the promotion of WAT inflammation during cachexia.

No MeSH data available.


Related in: MedlinePlus