NFκB and TNFα as individual key molecules associated with the cisplatin-resistance and radioresistance of lung cancer

Cisplatin-resistant (A549CisR and H292CisR) and radioresistant (A549R26 and H292R22) sub-line non-small cell lung cancer (NSCLC) cells were developed in our lab by long term treatment of parental cells with cisplatin or radiation. Our data showed no cross-resistance between these two sets of cell lines, indicating that molecular mechanisms of developing each resistance may be different. Using these sub-line cells, we sought to reveal the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells, compared with parental cells. In qPCR analyses of screening DNA repair and cell survival-associated molecules, we identified NFκB and TNFα as the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells, respectively, compared with parental cells. Western blot analysis of parental vs. resistant cells and the IHC staining of tumor tissues of A549P, A549CisR, and A549R26 cell-derived xenografts in mice confirmed such results. Next, studies using specific inhibitors of NFκB and TNFα and experiments using NFκB and TNFα-knocked down cells showed that inhibition or knockdown of NFκB overcame cisplatin-resistance, while inhibition or knockdown of TNFα increased radiosensitivity of radioresistant lung cancer cells. Therefore, these two molecules may be used as markers of the prognosis/diagnosis of individual resistance development during lung cancer treatment.     

IκBα polymorphisms were associated with increased risk of gastric cancer in a southern Chinese population: a case-control study

AimNuclear factor-kappa B inhibitor alpha (IκBα) polymorphisms were found to be associated with inflammatory diseases. However, the association between IκBα polymorphisms with gastric cancer is still unknown. We aim to investigate the association between IκBα polymorphisms and gastric cancer risk in a large population-based case–control study among southern Chinese.Main methodsA population-based case–control study was conducted between 1999 and 2006 in Guangdong Province, China. A total of 1010 gastric cancer patients and 1500 healthy controls were enrolled in this study. IκBα polymorphisms were identified by sequencing of IκBα gene ranging from the 2 kb promoter region to the 3.5 kb genomic region. Polymorphisms in IκBα were analyzed by TaqMan SNP genotyping assay.Key findingsrs17103265 deletion homozygote (?/?) had significantly increased gastric cancer risk (OR = 2.11, 95% CI = 1.17–3.83, P = 0.01), compared with rs17103265 T homozygote (TT). rs17103265 (?/?) genotype was significantly associated with increased risk of intestinal-type gastric cancer with (OR = 2.21, 95% CI = 1.19–4.08, P = 0.01), but not with the diffuse or mix type of gastric cancer. rs17103265 (?/?) was associated with poorly differentiated gastric cancer (OR = 2.05, 95% CI = 1.07–3.94, P = 0.03), but not with moderately or well differentiated gastric cancer. A significant decrease in luciferase activity was observed in rs17103265 deletion allele as compared with the vector containing the rs17103265 T allele (P < 0.0001). rs17103265 polymorphism was not associated with the prognosis of gastric cancer patients.SignificanceIκBα rs17103265 deletion homozygote is a novel genetic risk factor for gastric carcinogenesis, especially for the development of certain subtypes of gastric cancer in southern Chinese population.     

Functional polymorphism of cyclooxygenase-2 gene (G–765C) in chronic obstructive pulmonary disease patients

Cyclooxygenase two (COX-2) is an important enzyme metabolizing arachidonic acid. In contrast to constitutive cyclooxygenase one (COX-1), COX-2 is induced by proinflammatory factors. Polymorphism −765G/C in COX-2-encoding gene promoter is associated with development of Alzheimer’s disease, depression, carcinoma of the pancreas in smokers, breast cancer and rheumatoid arthritis. It is interesting whether the −765G/C polymorphism in COX-2-encoding gene promoter can be associated with COPD, a disease which is inflammatory in character. It is highly probable as the breast and pancreas cancers, whose associations with the analyzed polymorphism have been studied, are smoking-dependent tumors. Additionally, tobacco smoke has been demonstrated to induce COX-2 in the lungs. The study group consisted of 122 COPD patients (48 females, 74 males). The control group consisted of 149 healthy nonsmoking subjects (83 females, 66 males). Polymerase chain reaction/restriction fragment length polymorphism was used for genotyping. A statistically significant difference in genotype distribution was observed as a result of the comparison between healthy subjects and patients with COPD. The distribution of alleles in both groups conformed with Hardy–Weinberg equilibrium. In the group of COPD patients, GG allele was found in 79 subjects, GC in 36, and CC in 7 subjects (F = 0.094, P = 0.296927); in the control group, 73 subjects had GG allele, 68—GC and 8—CC (F = 0.12728, P = 0.120265). The allele frequency revealed differences between those groups, attaining the level of statistical significance (χ² = 29.043, df = 2, P = 0.0000. The carriers of −765G allele are at 1.53-fold higher risk of developing COPD. The presence of GG genotype does not increase significantly the risk of the disease. It is also noteworthy that the carriers of CC or GC genotypes are at significantly lower risk of developing COPD than the group of subjects with GG genotype.     

IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice

Background

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.

Methodology and Principal Findings

The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.

Conclusions and Significance

This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

Serum interleukin-1β as a marker for differentiation of asthma and chronic obstructive pulmonary disease

Asthma and chronic obstructive pulmonary disease (COPD) are diseases of airway inflammation with clinical and physiological similarities, making their differentiation difficult. Airway inflammatory changes are associated with systemic changes. However, no serum marker is known for their differentiation. Therefore, serum interleukin (IL)-1β levels were determined. Out of a total of 1023 patients screened, we included in the study ten patients each with atopic asthma, non-atopic asthma and COPD and ten healthy subjects. Skin prick tests with 14 inhalant allergens were performed on each patient. Blood was collected in the symptomatic and asymptomatic phases of the diseases and serum IL-1β and IgE levels were determined. Our results showed that in the symptomatic phase in asthmatics, serum IL-1β levels were higher (P<0.05) than in patients with COPD. Serum IgE levels were higher (P<0.05) in atopic asthmatics than in non-atopic asthmatics and in COPD patients. We conclude that serum IL-1β level determination during the symptomatic phase of the diseases may help to differentiate asthmatics from patients with COPD. Serum IgE levels may differentiate atopic asthmatics from non-atopic asthmatics and COPD patients.     

Inhibitor IκBα promoter functional polymorphisms in patients with multiple sclerosis

The aim of this study is to investigate the association of IκBα promoter polymorphisms with the development of Multiple Sclerosis (MS) disease in Iranian population. One hundred and fifty patients with MS along with 150 unrelated healthy controls were enrolled in this study. The IκBα ?881A/G (rs3138053), ?826C/T (rs2233406) and ?519C/T (rs2233408) polymorphisms were determined by the polymerase chain reaction/restriction fragment length polymorphism method. This study demonstrated that the genotype frequencies of IκBα ?881A/G and ?826T/T, and allele frequencies of IκBα?881G were significantly higher in patients with MS with respect to as compared to the controls. We also found that the estimated haplotype frequency of IκBα promoter ?881G–826T–519C was significantly increased in the patient with MS in comparison with that of the healthy individuals. This study reveals that polymorphisms in the IκBα promoter (?881 A/G, ?826 C/T) are strongly associated with the susceptibility of Iranian MS patients.     

Tumor Necrosis Factor-α +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease

Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs). Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.     

Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKβ, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKβ inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKβ, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.     

Enhanced expression of human β-defensin 2 in peripheral lungs of patients with chronic obstructive pulmonary disease

Human β-defensin 2 (hBD-2) has antimicrobial activity and may play a role in airway mucosal defense, but studies have not yet examined its expression in lung tissue of patients with chronic obstructive pulmonary disease (COPD). Here we investigated hBD-2 levels in lung tissues of COPD patients and analyzed their correlations with IL-8, IL-1β, cigarette smoking and lung function in order to see whether the protein may be involved in pathogenesis of the disease. Peripheral lung tissue specimens were obtained from 51 patients who underwent lung resection for peripheral lung cancer: healthy non-smokers (n = 8), healthy current smokers (n = 7), non-smokers with COPD (n = 11), and current smokers with COPD (n = 25). RT-PCR and immunohistochemical staining were used to detect expression levels of hBD-2, IL-8 and IL-1β. Expression of hBD-2 mRNA was significantly higher in COPD patients than in healthy controls, and significantly higher in current smokers than in non-smokers (p < 0.05). Among healthy controls, hBD-2 mRNA levels were similar between current smokers and non-smokers. Immunohistochemistry showed hBD-2 protein to be expressed mainly in epithelia of distal bronchioles and its expression pattern among our patient groups mirrored that of the mRNA. IL-8 mRNA levels were significantly higher in COPD patients than in healthy controls (p < 0.05), while IL-1β mRNA levels did not differ significantly among the groups. Levels of hBD-2 mRNA positively correlated with levels of IL-8 mRNA (r = 0.545, p = 0.002), and negatively correlated with FEV1/FVC ratios and with predicted FEV1% values (r = −0.406, p = 0.011). Our results indicate that hBD-2 expression is elevated in distal airways of COPD patients and that it may be involved in pathogenesis of the disease. Our data implicate cigarette smoking as a factor that may elevate hBD-2 levels in lung tissues of COPD patients.     

Tumor Necrosis Factor-α +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease

Background  

Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory process, in which the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α is considered to play a role. In the present study the putative involvement of TNF-α gene polymorphisms in pathogenesis of COPD was studied by analysis of four TNF-α gene polymorphisms in a Caucasian COPD population.     

Histamine regulates cyclooxygenase 2 gene activation through Orai1-mediated NFκB activation in lung cancer cells

Histamine, an important chemical mediator, has been shown to regulate inflammation and allergic responses. Stimulation of histamine receptors results in a significant increase in cytoplasmic Ca²⁺, which could be mediated by inositol trisphosphate (IP₃)-dependent store-operated Ca²⁺ channels (SOC). However, the link between histamine-mediated signaling and activation of inflammatory genes such as cyclooxygenase 2 (COX-2) is still unknown. Our study indicated that the COX-2 protein was highly expressed in human lung cancer cells. Following stimulation with 10 μM of histamine, both store-operated Ca²⁺ entry (SOCE) and COX-2 gene expression were evoked. Histamine-mediated COX-2 activation can be prevented by 2-APB and SKF-96365, SOC channel inhibitors. In addition, deletion analysis of the COX-2 promoter suggested that the region between −80 bp and −250 bp, which contains NFκB binding sites, is the key element for histamine-mediated signaling. Knocking down ORAI1, one of the essential molecules of store-operated calcium channels, attenuated histamine-mediated COX-2 expression and NFκB activation. These results indicated that ORAI1-mediated NFκB activation was an important signaling pathway, responsible for transmitting histamine signals that trigger inflammatory reactions.     

Dietary soy and tea mitigate chronic inflammation and prostate cancer via NFκB pathway in the Noble rat model

Chronic inflammation and nuclear factor-kappa B (NFκB) have been implicated in prostate cancer development; thus, dietary factors that inhibit NFκB may serve as effective chemo-preventative agents. Prostate cancer risk is significantly lower in Asian countries compared to the United States, which has prompted interest in the potential chemopreventative action of Asian dietary components such as soy and green tea. This study examined the effects of dietary soy and tea on NFκB activation and inflammation in vivo using a hormone-induced rat model for prostate cancer. Male Noble rats implanted with estradiol and testosterone were divided into 4 dietary groups: control, soy, tea, or soy+tea. NFκB activation and inflammatory cytokines were measured post implantation. The combination of soy and tea suppressed NFκB p50 binding activity and protein levels via induction of IκBα. Soy and tea also decreased prostate inflammatory infiltration, increased Bax/BcL2 ratio and decreased protein expression of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1β compared to control. Soy and tea attenuated prostate malignancy by decreasing prostate hyperplasia. These effects were not apparent in groups treated with soy or tea alone. The ongoing in vivo studies thus far suggest that combination of foods, such as soy and tea, may inhibit hormone-induced proinflammatory NFκB signals that contribute to prostate cancer development.     

Lipopolysaccharide-Induced NF-κB Activation in Human Endothelial Cells Involves Degradation of IκBα but Not IκBβ

We studied the signal transduction pathways involved in NF-κB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitorN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-κB DNA-binding activity induced by LPS treatment. LPS induced IκBα degradation at 30–60 min after treatment, but did not induce IκBβ degradation up to 120 min. In contrast, TNF-α rapidly induced IκBα degradation within 5 min and IκBβ degradation within 15 min. Cycloheximide did not prevent LPS-induced IκBα degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IκBα degradation. LPS-induced IκBα degradation was inhibited by ALLN, confirming that ALLN inhibits NF-κB activation by preventing IκBα degradation. Of note, HMA also inhibited LPS-induced IκBα degradation. However, tyrosine phosphorylation of IκBα itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IκBα is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-κB-dependent genes through degradation of IκBα, not IκBβ, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IκBα.