Abstract
OBJECTIVE To study the expression of the nuclear factor kappa B (NF-kappa B) in non-small cell lung cancer (NSCLC), to explore the apoptotic ratio in NSCLC related to different NF-kappa Bs, and to understand the clinical significance of NF-kappa B in NSCLC apoptosis.
METHODS NF-kappa B expression in 45 new samples of NSCLC, collected during a period from October to December, 2005, was assayed using Western blots, and the apoptotic ratio of NSCLC was determined by the Tunel method.
RESULTS Of the 45 patients, the average relative expression of NF-kappa B was 0.6047 ± 0.3572. The expression of NF-kappa B was higher in the poorly differentiated lung cancer cells than in the well-differentiated tumors (P < 0.05). The apoptotic ratio was 56.4% in the lung cancer cells with higher NF-kappa B expression, and was 76.7% in those with lower NF-kappa B expression (P < 0.05).
CONCLUSION The expression of NF-kappa B is correlated with the differentiation of NSCLC. NF-kappa B inhibits apoptosis in NSCLC. As a transcription factor, NF-kappa B plays a very important role both in formation and in development of NSCLC. NF-kappa B might serve as a target for NSCLC gene therapy.
keywords
Introduction
There is a close relationship between the transcription factor NFkappa B (nuclear factor kappa B) and regulation and control of cellular proliferation and tumorigenesis[1]. NF-kappa B belongs to the Rel family of transcription factors. In mammals, the Rel family is composed of the RelA/p65, C-Rel, RelB, P50 (NF-kappa B 1) and P52 (NF-kappa B 2). A typical feature of the NF-kappa B family is that all members share a common homologous highly conserved Rel region, which combines with DNA to form a dipolymer and interacts with the inhibitor (I-Kappa B)[2]. In resting cells, the NF-kappa B resides in the cytoplasm with a profilin (I-Kappa B) in an inactive form. I-Kappa B can activate the NF-kappa B when phosphorylated and is degraded by a proteolytic enzyme. This shifts the latter inhibitor into the cell nucleus, when it is integrated with the promotor and reinforcement region of a specific gene that controls expression of a specific gene. This results in induction of the immune and inflammatory response, as well as regulating apoptosis[3,4].
At present, the role of NF-kappa B in apoptosis is not clear, which might be used either to enhance[5,6] or depress apoptosis[7]. In our study, the Tunel method was used to investigate the effect and clinical significance of NF-kappa B on apoptosis in non-small cell lung cancers (NSCLC).
Patients and Methods
Patients
A total of 45 NSCLC patients who had undergone surgery in our hospital, during a period from October to December, 2005 were selected. There were 34 males and 11 females, with a mean age of 59.6 years. All tumors were solitary. The surgical procedures were successful, without other preoperative treatments. Histological classification was based on the WHO typing, and the TNM stages were determined in accordance with the UICC clinical classification of tumors. Part of the excised tumors and normal homolateral pulmonary tissues were taken for frozen sections.
Methods
Western blots
Normal, paraneoplastic pulmonary tissues and lung cancers were separately divided, and radioimmune precipitation assay (RIPA) buffer was added, followed by incubation in ice. The Coomassie brilliant blue method was used to determine the protein concentration and a 50 μg protein sample was employed for electrophoretic separation by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), followed by a damp-dry transmembrane (provided by the Biokad Co. Ltd., USA) transfer. The first antibody (rabbit-antihuman, bought from the Santa Cruz Co. Ltd., USA, with a dilution of 1 vs. 500) and the second antibody (sheep-antirabbit from the Santa Cruz Co., with a Dilution of 1 vs. 5000) were respectively added followed by an enhanced chemiluminescence (ECL) reaction. Bandleader software was used to analyze the specific protein level of each electrophoretic channel and of β-actin, and to calculate the relative protein amounts.
Tunel method
The Tunel kits were purchased from the Roche Pharmaceutical Factory. Frozen 5 μm lung-cancer sections were fixed in 4% paraform aldehyde. Then a solution of 0.3% of H2O2 formaldehyde was applied for 30 min to block the endogenous peroxidase, and to conduct PBS washing. Tritonx-100 was added, and a Tunel mixed solution reaction was performed, with the addition of the POD transforming agent. Finally DAB stain was added and the sections mounted. Apoptotic nuclei were stained with a brownish-yellow color, and by examination of 1,000 cells in 3 different fields of vision, the positive cells were counted. An apoptotic index (AI) of over 20% was defined as positive. The results were determined by two medical doctors.
Statistical analysis
The t-test was employed to compare the statistical analysis of the data. A P < 0.05 was considered to show a significant difference in the apoptotic rate of various NF-kappa B expressions.
Results
The mean value of the NF-kappa B’s relative amount was 0.6047 ± 0.3572 (Fig.1), which was not related to the patients’ age, gender, stages, size and site of the tumor, or pathological type, P > 0.05. There was a high expression of NF-kappa B in the poorly-differentiated lung cancer cells (28 cases), and a low expression in the well-differentiated lung cancer cells (17 cases), t = 3.640, P = 0.001 (Table 1).
The β-actin expression in lung cancer and normal paraneoplastic tissues.
Clinical features of NSCLC and expression of NF-kappa B.
In the lung cancers with a high relative expression of NF-kappa B (0.6047 ± 0.3572, n = 27), the Tunel method was used to detect apoptosis in the cases among which 21 were positive and 6 negative for apoptosis. There was a low apoptotic rate (44.4%) in the lung cancer cells. In the lung cancer cases with a low expression of NF-kappaB (n = 18), 8 were positive and 10 negative, with an apoptotic rate of 77.8%, χ2 = 5.237, P = 0.022 (Figs.2 and 3).
Positive expression of NF-kappaB in the Tunel assay (× 400).
Negative expression of NF-kappaB in Tunel (× 400).
Discussion
Stark et al.[5] have reported concentration-dependent apoptosis in the colon cancer SW480 cell line treated with aspirin. The electrophoretic mobility shift assay (EMSA) showed that with a concentration-dependent increase of NF-kappaB (P50/P65) DNA binding, aspirin induced I-KappaB 2 degradation and programmed cell death. The NF-kappaB nuclear transport inhibitor hampered aspirin-induced apoptosis. A study by Chern et al.[6] showed that folate deficiency-induced apoptosis initiated with enhancement of NF-kappaB activity.
However recent research has shown that NF-kappaB plays an important role in protecting apoptosis. Rahman et al.[7] found that in breast cancer cell lines, inactivation of NF-kappaB was involved in regulation of I-3-C (indole-3-methanol) induced apoptosis. Choi et al.[8] discovered that β-lapachone can increase growth inhibition and induction of apoptosis in a concentration-dependent manner in the human colon HCT-116 cancer cell line. After treatment with β-lapachone, the NF-kappaB protein level and NF-kappaB-DNA binding significantly decreased. The effect of β-lapachone might also cause depression of the activity of NF-kappaB luciferase reporter plasmid, suggesting that the β-lapachone-induced apoptosis could be regulated by inactivation of NF-kappaB. The data from our study indicated that in NSCLC, the high expression of NF-kappaB can inhibit apoptosis in lung cancer cells.
The nuclear factor NF-kappaB has found to be activated in many kinds of human tumors, thus facilitating canceration of the cells, as the nuclear factor can prevent apoptosis. Denlinger et al.[9] pointed out that in most NSCLC cases, NF-kappaB was involved in non-conventional anti-apoptosis. Jones et al.[10] showed that in all NSCLC cell lines, chemotherapy induced NF-kappaB nuclear translocation and binding with DNA. After chemotherapy, NF-kappaB was needed for survival of the cells. Stage-I clinical trials have demonstrated that inhibition of NF-kappaB activity can significnatly reduce the role of anti-apoptosis in NSCLC, and can strengthen the sensitivity of chemotherapy.
The data from our study indicated that the expression of NF-kappaB is closely correlated to poorly-differentiated NSCLC, which might also be one of the reasons for NSCLC tolerance to chemotherapy and poor prognosis. To clarify the drug tolerance and signaling transduction pathway of NF-kappa B, will increase the sensitivity of NSCLC to chemotherapy, and improve the prognosis of the patients. Therefore, chemotherapy in combination with studies on inhibition of dependent-cell survival may become a clinical strategy for treatment of advanced NSCLC.
Footnotes
This work was supported by a grant from Educational Commission of Heilongjiang Province, China (No.115113049).
- Received August 16, 2007.
- Accepted December 18, 2007.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.