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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 3  |  Page : 516-520

Nuclear factor-kappa B as potential therapeutic target in human colon cancer


1 Department of Cytophysiology, Chair of Histology and Embryology, Medical University of Silesia, Medyków 18, Katowice, Poland
2 Department of Proteomics, Medical University of Silesia, 41-200 Sosnowiec, Poland

Date of Web Publication12-Jun-2018

Correspondence Address:
Dr. Danuta Plewka
Department of Cytophysiology, Chair of Histology and Embryology, Medical University of Silesia, Medyków 18, 40-752 Katowice
Poland
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.180607

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 > Abstract 


Background: It has been proved that nuclear factor-kappa B (NF-κB) is activated in all cells, promotes proliferation of cells, regulates the immunological and inflammatory response, and contribute to the pathogenesis of many conditions, including cancer. Many studies pointed to constitutive activation of NF-κB in cells of certain malignant tumors.
Objective: The aim of the study was to analyze the role of nuclear growth factor κB as colon cancer marker and prognostic factor.
Materials and Methods: The study included 59 primary colorectal tumor patients and 15 patients in control group. The tumor samples were taken during partial colectomy and colonoscopy in control group. Tissues samples were fixed and embedded in paraffin blocks and cut. Sections were used for schedule immunohistochemical staining with the application of specific antibody for NF-κB epitope. The marker expression was compared with well-known prognostic factors in colon tumors such as tumor type, stage, and grade to establish if it might be a potential prognostic factor.
Results: The results showed statistically significant difference between control group and cancer group.
Conclusions: The expression NF-κB did not depend on the stage and grade of colon tumors.

Keywords: Colon cancer, nuclear factor-kappa B, stages of neoplastic differentiation


How to cite this article:
Plewka D, Plewka A, Miskiewicz A, Morek M, Bogunia E. Nuclear factor-kappa B as potential therapeutic target in human colon cancer. J Can Res Ther 2018;14:516-20

How to cite this URL:
Plewka D, Plewka A, Miskiewicz A, Morek M, Bogunia E. Nuclear factor-kappa B as potential therapeutic target in human colon cancer. J Can Res Ther [serial online] 2018 [cited 2020 Jul 8];14:516-20. Available from: http://www.cancerjournal.net/text.asp?2018/14/3/516/180607




 > Introduction Top


Epidemiological studies revealed that chronic inflammatory conditions make the patients more susceptible to different carcinomas. Factors triggering a chronic inflammatory condition, which increases the risk of developing cancer, include micro-organism infections, auto-immune diseases as well as cryptogenic inflammatory conditions.

A number of proinflammatory factors, such as tumor necrosis factor-alpha, lipopolysaccharides or a variety of drugs, may activate nuclear factor-kappa B (NF-κB), mainly through kinase (IκB kinase [IKK]-dependent phosphorylation and degradation of inhibitory proteins IκB.

It has been proved that NF-κB is activated in all cells to regulate the expression of different target genes which promote proliferation of cells, regulate the immunological and inflammatory response, and contribute to the pathogenesis of many conditions, including cancer.[1],[2],[3] Many studies pointed to constitutive activation of NF-κB in cells of certain malignant tumors, such as lymphoma, leukemia, breast tumors, melanoma, pancreatic, or colorectal cancer.[4] Many of the reports suggest that constitutively active NF-κB is associated with negative properties of neoplastic cells, such as anti-apoptotic effect, cell growth, and metastasis.[5] It should be remembered; however, that tumor cells with constitutively active NF-κB have not been closely studied. Moreover, effects of such constitutive activation in vivo are not well recognized.

Probable relations between inflammation and cancer was suggested by Virchow, who hypothesized that malignant tumor develops in the regions of chronic inflammation, understanding that different “irritating factors” cause damage of tissue, trigger inflammation, and promote proliferation of cells.[6],[7] It has been established that more than 15% of all malignancy cases are initiated by chronic inflammatory conditions.[8],[9],[10],[11],[12] Although the exact inflammatory mechanism initiating cancer has not been fully recognized, it is known that NF-κB may appear as an important contributory factor as it is activated in chronic inflammation and its constitutive activation occurs in many known tumors.

It has been shown that NF-κB regulates a number of genes included in the process of cell proliferation, neoplasia, and metastasis. Cyclin D1 and cMyc, two target genes for NF-κB, take an important part in cell growth and proliferation,[13] as well as key factors of angiogenesis, such as the vascular endothelial growth factor and interleukin-8 (IL-8)[14] and are directly or intermediately strengthened by NF-κB activation. Inhibition of colon cancer growth by inhibition of NF-κB has been shown earlier.[15]

The key role of NF-κB signaling pathway in apoptosis, promotion and maintenance of tumor, suggests explicitly that NF-κB inhibitors will prove helpful in cancer therapies. Much effort is now being put to develop NF-κB inhibitors to fight cancer.[4],[16],[17] Proteasome inhibitors regulate degradation of IκB inhibiting therefore NF-κB; several laboratories proved their efficacy in expression of apoptosis along the cancerous cells lines, together with chemotherapeutic drugs, or cytokines inducing cell death.[18],[19] Sulphasalazine and methotrexate are commonly used in the treatment of acute inflammatory bowel disease conditions through inhibition of NF-κB activation;[20],[21] therefore, they are likely to reduce the risk of developing cancer.

As activation of NF-κB has a role in anti-apoptotic processes, cell growth, metastasis, and expression of chemokines, inhibition of NF-κB activation may appear helpful in cancer therapies. Indeed, there are several reports on NF-κB inhibition as a therapeutic method in cancer,[22] which however are insufficient to accept NF-κB inhibition as the first line cancer therapy. The aim of this study was indication how grading (G1–G4) colorectal cancers effects on NF-κB expression?


 > Materials and Methods Top


The study included 59 primary colorectal tumor patients. The median age was 61.5 years (range: 41–74). The other clinicopathological characteristics are listed in [Table 1]. The control group (15 patients: Seven women, eight men, and median age: 51 years) included the large intestine samples, taken for reasons other than the conditions specified above. The group included the hospitalized patients in whom colonoscopy showed normal intestine or only some single polyps.
Table 1: Patient characteristics

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Immunohistochemical tests

Colon samples were fixed in 4% paraformaldehyde for 24 h at 4°C and then dehydrated, cleared in xylenes, and embedded in paraffin. The samples were sectioned into 5 μm sections.

Paraffin sections were mounted on silane-coated slides, dewaxed, and rehydrated through a decreasing series of alcohol. The sections were treated with 10 mM citrate buffer, pH 6.0 in water bath (30 min at 95°C) for antigen retrieval, then treated with 3,0% (v/v) H2O2 (dissolved in methanol) for 10 min for quenching of endogenous peroxidase activity, and washed in 10 mM phosphate buffered saline (PBS) - 0.05% v/v Tween 20, pH 7.5. Nonspecific binding was reduced by incubation in normal goat serum for 60 min. Then, slides were incubated with rabbit anti-NF-κB p50 (Santa Cruz Biotechnology, CA, USA, sc-114, dilution 1/200) polyclonal antibody in a humidified chamber for 22 h at 4°C. After washing in PBS-Tween 20, the sections were incubated with biotinylated goat anti-rabbit immunoglobulins (Vector Laboratories, Burlingame, CA, USA) for 30 min, and next with avidin/biotinylated peroxidase complex (Vector) for 30 min. The bound antibodies were visualized with diaminobenzidine (Vector) in PBS, pH 7.5 according to the manufacturer's instructions. Finally, the tissues were counterstained with Gill's hematoxylin, dehydrated, and coverslipped. Negative control was provided by sections where the primary antibody was replaced by rabbit IgG, at solution such as the primary antibody. The control was performed in parallel for each slide to reveal the nonspecific binding of the primary antibody.

Quantitative analysis

Quantitative analysis was performed for the evaluated proteins in the sections tested. With the use of NIS-Elements Advanced Research 4.00.12 (Nikon, Tokyo, Japan), the optical density of microscope preparations was evaluated in areas showing the color reaction to a selected protein.

The immunohistochemically stained slides were examined under an Eclipse E200 microscope with DS-Fi1 digital camera (Nikon, Tokyo, Japan). Microscopy images were obtained at ×200 magnification.

The images were analyzed using the software NIS-AR (Nikon). In each positively stained cell, the intensity of staining was measured as the optical density of the reaction product.

Three sections from each patient were counted to obtain a mean value of positive labeling per se ction. An average optical density was calculated.

Statistical analysis

The results were collected in the database. The sum optical density of the immunohistochemical reaction for NF-κB product was studied in five groups: Control and colorectal cancers in four grades (G1, G2, G3, and G4). Measured values were analyzed with the one-way analysis of variance. For multiple comparisons post hoc, Turkey test was used. Assessment of the data normal distribution based on the Shapiro–Wilk test. Homogeneity of variance was rated by Levene test. Values were presented as arithmetic means ± standard deviation. The differences were considered statistically significant at significance level P < 0.05. The statistical analysis was performed with Statistica 10 (StatSoft, Tulsa, OK, USA).


 > Results Top


Control

Expression of this factor was poorly manifested in assays representing the control study. It has been shown that expression of the protein was manifested in individual stromal cells and partly in colon crypt cells [Figure 1]. No expression of this factor was observed in enterocytes or mucocytes.
Figure 1: Immunohistochemical localization of nuclear factor-kappa B at particular stages of cell differentiation in colorectal cancer (×200)

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Colorectal tumor

Grade G1

Expression of the NF-κB in the studied tumor of a large intestine showed very high optical density of the product of immunohistochemical reaction. It was 5-fold higher than control group [Figure 2]. Expression of the protein was shown in the stromal and tumor cells [Figure 1].
Figure 2: Optical density of the reaction product for nuclear factor-kappa B at different cell differentiation grades in human, colorectal tumors. *Refer to statistically significant changes for P < 0.05 between the control values and the cancer grade

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Grade G2

NF-κB expression was slightly higher than observed at Grade G1 [Figure 2]. Expression of the factor was shown only in part of the adenocarcinoma cells [Figure 1].

Grade G3

Expression of NF-κB was similar to that revealed at Grade G1 [Figure 2] and was observed in both cancer and stromal cells [Figure 1].

Grade G4

Expression of NF-κB at Grade G4 was the lowest in all grade groups but higher than control group [Figure 2]. Reaction to this factor was observed in both cancer and stromal cells [Figure 1].

Comparison

Expression of this NF in colorectal tumors was markedly higher than the control values. It was shown to be 5-fold higher at particular grades of colorectal cancer. However, no statistically significant differences were noted between the individual grades.


 > Discussion Top


Colorectal cancer is a life-threatening complication of intestinal inflammatory conditions, where NF-κB may contribute to the mechanical association of inflammation with neoplasia. The molecular mechanisms, underlying such process, have recently been more explicitly recognized, throughout the performed biochemical and genetic studies. We have already defined NF-κB signaling pathway, which may be accepted as a rational objective of cancer therapy.[3],[23],[24] Recently, great expectations have aroused, following experiments with cell cultures and animal models, which had focused on the use of NF-κB inhibitors as a new cancer therapy.[25],[26],[27] However, subsequent experimental results failed to establish NF-κB as a common molecular target in the different cancers. For example, IKKβ inhibitors proved to inhibit phosphorylation of IκB more specifically while they inhibit activation of NF-κB only in part and seems to offer a limited application.[12] Specific roles of different NF-κB subunits in particular types of cancer, with a special focus on the biological behavior of malignant forms, such as survival, proliferation, angiogenesis, differentiation and metastasis, and demand further studies. The relation between inflammation and cancer has now merely found an overall acceptance. However, the further question is asked whether preventing inflammation is the best mode of a cancer therapy. An ideal NF-κB inhibitor should prevent NF-κB activation with no apparent effects along other signaling pathways and should avoid prolonged immunosuppression.

It has been known for some time, now, that constitutive activation of NF-κB is often observed in cancer, including solid tumors, such as prostate, colorectal, or pancreatic cancer.[28] Such activation in cancer cells may affect an overexpression of a number of NF-κB targets genes, associated with the tumor growth, for example, anti-apoptotic genes, or those promoting the cell cycle. Moreover, constitutive activation of NF-κB has a role in resistance to chemotherapy and radiotherapy.

The levels of several chemokines associated with angiogenesis were elevated in cells with constitutive activation of NF-κB.[29],[30],[31] Making use of the colorectal tumor specimens from surgical resection as well as of the adjacent healthy tissues, it was shown that the levels of IL-8 and monocyte chemoattractant protein-1 were significantly higher in many neoplastic specimens. It should be remembered however that NF-κB inhibition alone is sufficient to inhibit angiogenesis accomplished through cancer cells. In such respect, NF-κB inhibition may appear as a powerful form of anti-angiogenic therapy in colorectal cancer. Moreover, NF-κB activation is also the main regulatory step in anti-apoptosis.[3] It has also been proved that expression levels of genes associated with apoptosis, such as A20 and cIAP2, were reduced by inhibition of constitutive activation of NF-κB while genes also associated with apoptosis, such as cIAP1 and CYLD, known to be regulated by NF-κB activation, were not visibly modified.[32]

Application of NF-κB inhibitor may prove helpful as a cancer therapy. Much of the research effort is now being devoted to the development of IKKβ/NF-κB inhibitors.[33] Test of NF-κB inhibitors for their possible clinical use showed that NF-kappa B essential modulator (NEMO) inhibitor blocks the binding of NEMO with IKKβ,[34] impairs NF-κB activity and reduces damage associated with the inflammatory condition, and the number of tumors associated with colitis.[35] This shows that blocking of NF-κB, aimed at IKKβ, with the use of peptide binding NEMO domains, may appear as a reasonable therapeutic approach in colitis and cancer associated with the inflammation. It is likely therefore that NF-κB inhibitor will prove effective only if combined with other anti-cancer measures or radiotherapy.

To sum up, constitutive activation of NF-κB is often observed in colorectal cancer, having major role in angiogenesis and anti-apoptosis and promoting the tumor growth. Molecularly targeted therapy against NF-κB activation should prove effective in colorectal cancer, in particular that with constitutive activation of NF-κB.


 > Conclusions Top


Expression of the inactive form of NF-κB in cytoplasm did not depend on the colorectal cancer stage.

Financial support and sponsorship

The study was planned according to statement of Human and Animal Rights. It has been approved by the Bioethical Committee of the Medical University of Silesia (no. KNW/0022/KB/55/10).

Conflicts of interest

There are no conflicts of interest.



 
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