Nuclear factor‐κB as a potential therapeutic target for the novel cytotoxic agent LC‐1 in acute myeloid leukaemia

Nuclear factor‐κB (NF‐κB) has been implicated in a number of malignancies and has been suggested to be a potential molecular target in the treatment of leukaemia. This study demonstrated the constitutive activation of NF‐κB in human myeloid blasts and a clear correlation between NF‐κB expression and in vitro cytoprotection. High NF‐κB expression was found in many of the poor prognostic acute myeloid leukaemia (AML) subtypes, such as French‐American‐British classification M0 and M7, and the poor cytogenetic risk group. The in vitro effects of LC‐1, a novel dimethylamino‐parthenolide analogue, were assessed in 62 primary untreated AML samples. LC‐1 was found to be cytotoxic to AML cells in a dose‐dependent manner, mediated through the induction of apoptosis. The median drug concentration necessary to kill 50% of the cells was 4·5 μmol/l for AML cells, compared with 12·8 μmol/l for normal marrow cells. LC‐1 was shown to reduce the five individual human NF‐κB Rel proteins in a dose‐dependent manner. The subsequent inhibition of many NF‐κB‐regulated cytokines was also demonstrated. Importantly, sensitivity to LC‐1 was correlated with the basal NF‐κB activity. Consequently, LC‐1 treatment provides a proof of principle for the use of NF‐κB inhibitors in the treatment of AML.     

Role of definitive chemoradiotherapy using docetaxel and 5‐fluorouracil in patients with unresectable locally advanced esophageal squamous cell carcinoma: a phase II study

Definitive chemoradiotherapy (CRT) with docetaxel (DOC) and 5‐fluorouracil (5‐FU) is a unique regimen for esophageal cancer. In this prospective phase II study, antitumor effect and safety of CRT using DOC and 5‐FU for inoperable locally advanced esophageal cancer were evaluated. DOC 7.5 mg/m² was infused on days 1, 8, 22, and 29. 5‐FU 250 mg/m²/day was infused continuously on days 1–5, 8–12, 15–19, 22–26, 29–33, 36–40, and 43–45. Radiotherapy was given to 66 Gy in 33 fractions. Eleven patients with thoracic and five with cervical esophageal cancer were eligible. All patients had esophageal squamous cell carcinoma (ESCC). The response rate was 94%, with complete response in five patients (31%) and partial response in 10 (63%). Hematologic toxicity was mild; only one patient (6%) had Grade 1 leukopenia. Nonhematologic Grade 3 or higher adverse events were esophagitis (31%), anorexia (6%), and esophago‐bronchial fistula (6%). No treatment‐related deaths occurred. The median time to progression was 20 months and overall 3‐year and 5‐year survival were 44% and 31%, respectively. Definitive CRT using DOC and 5‐FU could be performed safely, and it demonstrated a favorable antitumor effect for ESCC. This regimen might be indicated in patients in whom it is desirable to avoid myelosuppression and progression of renal impairment.     

Melatonin synergizes the chemotherapeutic effect of 5‐fluorouracil in colon cancer by suppressing PI3K/AKT and NF‐κB/iNOS signaling pathways

5‐Fluorouracil (5‐FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5‐FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5‐FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5‐FU. We found that melatonin significantly enhanced the 5‐FU‐mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5‐FU to promote the activation of the caspase/PARP‐dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5‐FU by targeting the PI3K/AKT and NF‐κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5‐FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF‐κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K‐ or iNOS‐specific inhibitor synergized the antitumor effects of 5‐FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5‐FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5‐FU in colon cancer through simultaneous suppression of multiple signaling pathways.     

Therapy-induced expression of NF-κB portends poor prognosis in patients with localized esophageal cancer undergoing preoperative chemoradiation

Activated nuclear factor‐kappa B (NF‐κB) in the pretreatment cancer tissue of patients with localized esophageal adenocarcinoma (LEA) undergoing preoperative chemoradiation is associated with poor prognosis. It is known that constitutively activated NF‐κB prior to any therapy portends poor prognosis, and it is also known that activated NF‐κB in the treated specimen is associated with poor prognosis. However, the prognosis of patients who have treatment‐induced activation of NF‐κB (meaning their cancers activate NF‐κB during or after therapy) is not been reported. We hypothesized that the treatment‐induced activation of NF‐κB would impart poor prognosis similar to that imparted by constitutively activated NF‐κB cancer. Patients with LEA who had undergone preoperative chemoradiation plus surgery and had pre‐ and post‐therapy cancer tissue available were selected. Pre‐ and post‐therapy cancer tissues were stained by immunohistochemistry for nuclear staining of NF‐κB. The overall survival (OS) and disease‐free survival were assessed and compared for patients who had intrinsic constitutively activated NF‐κB cancer with those who had induced activation of NF‐κB only post‐therapy. A total of 41 patients with LEA were investigated. Twenty‐five patients had NF‐κB positive cancer at baseline, and 16 had NF‐κB negative cancer at baseline but became positive post‐therapy. There was no difference in the location, histology grade, clinical stage, or the curative resection (RO) resection rate in the two populations. OS (P = 0.71), disease‐free survival (P = 0.86), and median survivals (Converters: 24 months [95% confidence intervals: 7.78 to 40.22]vs. Nonconverters: 34.13 months [95% confidence intervals: 3.54 to 64.27]) were not different between the two groups. Our data suggest that activation of NF‐κB in response to stress/injury of therapy leads to poor OS. These results need to be confirmed in a larger number of patients. It may be that only pre‐therapy evaluation of NF‐κB is insufficient to assess prognosis of patients with LEA. Additional implications include that when effective anti‐NF‐κB therapies become available, they may have to be considered in patients whose cancers do not have constitutively activated NF‐κB or cancer may have to be monitored during therapy with biomarker assessments.     

Squamous cell carcinoma in Barrett's esophagus: field effect versus metastasis

Barrett's esophagus (BE) is a premalignant condition with an increased risk of developing esophageal adenocarcinoma (EAC). Risk factors for EAC overlap with those for esophageal squamous cell carcinoma (ESCC), but ESCC is surprisingly rare in BE. We report two cases of ESCC directly surrounded by BE. Both patients had a previous medical history of cancers, i.e., head and neck squamous cell carcinomas, and were using alcohol and smoking tobacco. Using immunohistochemistry for p63, CK5, CK7, and CDX2, it was confirmed that these carcinomas were pure squamous cell carcinomas, and not EACs or esophageal adenosquamous carcinomas arising from BE. Using TP53 mutation and loss of heterozygosity analysis, we established that the ESCCs in BE were not metastases of the previously diagnosed head and neck squamous cell carcinomas but de novo primary ESCCs. This study shows the strength of molecular analysis as an adjunct to the histopathologic diagnosis for distinguishing between metastases of prior cancers and primary cancers. Furthermore, these cases imply that presence of BE is not protective with regards to developing ESCC in the lower one third of the esophagus. We suggest that their ESCCs arose from islets of squamous epithelium in BE.     

Autoantibody detection to tumor‐associated antigens of P53, IMP1, P16, cyclin B1, P62, C‐myc,Survivn, and Koc for the screening of high‐risk subjects and early detection of esophageal squamous cell carcinoma

The aim of this study was to evaluate the diagnostic values by detecting sera autoantibodies to eight tumor‐associated antigens (TAAs) of P53, IMP1, P16, cyclin B1, P62, C‐myc, Survivn and Koc full‐length recombinant proteins for the screening of high‐risk subjects and early detection of esophageal squamous cell carcinoma (ESCC). Enzyme‐linked immunosorbent assay was used to detect autoantibodies against the eight selected TAAs in 567 sera samples from four groups, including 200 individuals with normal esophageal epithelia (NOR), 214 patients with esophageal basal cell hyperplasia (BCH), 65 patients with esophageal dysplasia (DYS), and 88 patients with ESCC. In addition, the expression of the eight antigens in esophageal tissues was analyzed by immunohistochemistry. Statistically significant distribution differences were identified among the four groups for each of the individual autoantibodies to six TAAs (P53, IMP1, P16, cyclin B1, P62, and C‐myc); the detection rates of antoantibodies were positively correlated with the progression of ESCC. When autoantibody assay successively accumulated to six TAAs (P53, IMP1, P16, cyclin B1, P62, and C‐myc), a stepwise increased detection frequency of autoantibodies was found in the four sera groups (6% in NOR, 18% in BCH, 38% in DYS, and 64% in ESCC, respectively), the risks to BHC, DYS, and ESCC steadily increased about 3‐, 9‐, and 27‐folds. The sensitivity and the specificity for autoantibodies against the six TAAs in diagnosing ESCC reached up to 64% and 94%, respectively. The area under the receiver operating characteristic curve for the six anti‐TAA autoantibodies was 0.78 (95% confidence interval 0.74–0.83). No more increasing in sensitivity was found with the addition of new anti‐TAA autoantibodies. A combination detection of autoantibodies to TAAs might distinguish ESCC patients from normal individuals and the patients with esophageal precancerous lesions.     

Overexpression of matrix metalloproteinase 10 is associated with poor survival in patients with early stage of esophageal squamous cell carcinoma

Matrix metallopeptidase 10 (MMP10) is frequently expressed and correlates closely with metastasis and poor prognosis in various human cancers. However, the significance of MMP10 expression in esophageal squamous cell carcinoma (ESCC) and its role in ESCC progression remains unclear. In this report, upregulation of MMP10 mRNA was detected in 39/60 (65.0%) of primary ESCC tissues compared with their paired nontumor esophageal tissues. Tissue microarray (TMA) study found protein overexpression of MMP10 in 188/239 (78.7%) of primary ESCC tissues but not in their corresponding nontumor esophageal tissues, suggesting that overexpression of MMP10 may play important roles in ESCC development and progression. Although the overexpression of MMP10 was not significantly associated with disease-specific survival rate (P= 0.182) for all tested ESCCs, it was significantly associated with poorer disease-specific survival (P= 0.001) in early stage of ESCCs (I-IIA). In addition, multivariate analysis found that MMP10 expression in tumor tissues was evaluated as a potential independent prognostic factor for early stage ESCC patients. These findings suggest that MMP10 plays an important role in ESCC progression in the early stage, and overexpression of MMP10 in tumor tissues could be used as a potential prognostic marker for patients with early clinical stage of ESCC.     

NF‐κB protects Behçet's disease T cells against CD95‐induced apoptosis up‐regulating antiapoptotic proteins

Objective

To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up‐regulation of antiapoptotic factors.

Methods

The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF‐κB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down‐regulate NF‐κB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF‐κB small interfering RNA.

Results

Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95‐induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up‐regulation of the short form of cellular FLIP (cFLIP) and Bcl‐x_L in BD activated T cells, while levels of Bcl‐2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IκB was up‐regulated, whereas NF‐κB translocated to the nucleus in BD T cells, suggesting that NF‐κB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF‐κB small interfering RNA down‐regulated cFLIP and Bcl‐x_L expression levels and sensitized BD activated T cells to CD95‐induced apoptosis.

Conclusion

Taken together, these results indicate that NF‐κB contributes to the regulation of the apoptosis‐related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF‐κB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune‐mediated disease.

     

Clinicopathological significance of nuclear factor‐κB activation in hepatocellular carcinoma

Aim: Nuclear factor‐κB (NF‐κB) is a critical signaling mediator in inflammation, apoptosis resistance and oncogenesis. It has been reported that NF‐κB is activated in several cancers, including hepatocellular carcinoma (HCC). Studies of genetic disruptions in mice also suggest that NF‐κB plays critical roles in hepatocarcinogenesis. The aim of the present study is to characterize NF‐κB activation and correlate it with the degree of malignancy in HCC. Methods: To examine the correlation between the positivity of the nuclear p50 subunit and HCC recurrence, we analyzed immunostaining of the NF‐κB p50 subunit in two groups of HCC samples with known prognosis and Akt phosphorylation status: 49 patients showing early recurrence within 6 months (group A) and 50 patients who were recurrence‐free for at least for 3 years (group B). Results: In group A, positive nuclear staining of p50 was shown in 18 cases (36.7%), whereas only one case (2.0%) in group B had positive nuclear staining of p50 (P = 2.48839 × 10^–5). This suggests a positive relationship between nuclear p50 and early recurrence and advanced HCC in humans. The presence of phosphorylated Akt correlated with nuclear staining of p50 in HCCs in group A (R² = 0.213, P < 0.001). Conclusion: Our results indicate that nuclear staining of p50 was clearly associated with early recurrent HCC, and the Akt pathway might play a role in NF‐κB activation in a subset of early recurrent HCC.     

Original article: Upregulation of caspase‐3 expression in esophageal cancer correlates with favorable prognosis: an immunohistochemical study from a high incidence area in northern China

Caspase‐3 plays an important role as the key effector during apoptosis, but there are very few studies of caspase‐3 in esophageal squamous cell carcinoma (ESCC). The purpose of this study was to investigate the expression and prognostic significance of caspase‐3 in ESCC from Linzhou City, a high incidence area in northern China. All 64 patients underwent esophagectomy for ESCC between January 2002 and December were enrolled in this study. Caspase‐3 expression was assessed by immunohistochemistry (IHC) in primary ESCC and paired normal esophageal epithelium. The positive rate of caspase‐3 expression was higher in ESCC than in normal esophageal epithelium (79.7% vs. 50.0%, Chi‐square = 12.372, P= 0.001). Caspase‐3 expression was correlated with tumor cell differentiation (Phi = 0.717, P < 0.001), tumor infiltration depth (Phi =?0.334, P= 0.008), and pathologic TNM (pTNM) staging (rs =?0.268, P= 0.032). Patients in caspase‐3 positive group had a significantly better 5‐year overall survival than those in the negative group (77.4% vs. 35.9%, χ²= 7.344, P= 0.007). Our results showed that caspase‐3 expression was upregulated in ESCC compared with normal esophageal epithelium in population of Chinese high incidence area, and patients with caspase‐3 positive expression had better prognosis. Therefore, caspase‐3 immunostaining could be a simple and useful tool for predicting survival in ESCC patients.     

Overexpression of cyclooxygenase‐2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients

Our objective was to investigate whether cyclooxygenase‐2 (COX‐2) expression can predict the patient's response to chemoradiotherapy (CRT) and ensuing prognosis in esophageal squamous cell carcinoma (ESCC). The clinicopathological and follow‐up data of 112 patients with ESCC who underwent CRT from January 2001 to June 2006 were analyzed retrospectively. The immunohistochemical expression level of COX‐2 was examined for all biopsy specimens of primary tumors, and the correlation of COX‐2 expression with the patient's response to CRT and prognosis was examined. COX‐2 positive immunostaining was detected in 111 (99.1%) of the patients, including overexpression in 54 (48.2%) patients and low expression in 58 (51.8%) of the patients. The response of tumors with a low level expression of COX‐2 (70.7%, 41/58) was significantly higher than that of tumors with COX‐2 overexpression (42.6%, 23/54; P = 0.003). Patients with a low level of COX‐2 expression had a higher downstaged rate than those with a high level of COX‐2 expression (9/13 vs 2/8), but the difference was not statistically significant (P = 0.08). In the definitive CRT group (91 cases), COX‐2 overexpression was significantly associated with poor 3‐year overall survival (P = 0.028). Multivariate analysis showed that only metastatic stage (nonregional node metastasis) was an independent prognosis factor. The assessment of COX‐2 status may provide additional information to identify ESCC patients with poor chances of response to CRT and potential candidates for more individualized treatment.     

Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Pilot study special AT‐rich sequence‐binding protein 1 investigating as a potential biomarker for esophageal squamous cell carcinoma

In the present study, we aimed to evaluate the expression of special AT‐rich sequence‐binding protein 1 (SATB1) in esophageal squamous cell carcinoma (ESCC) and assess the correlation between its expression and the clinicopathological features and prognosis of the disease. SATB1 expression in ESCC tissue was determined by using immunohistochemical analysis, quantitative real‐time polymerase chain reaction, and western blot analysis. The relationship between SATB1 expression and clinicopathological features was examined by using the chi‐squared test, and the survival rate was calculated by using the Kaplan–Meier survival curve. The correlation between the indicators and patient survival was estimated by using a Cox regression analysis. High SATB1 expression in was detected in 48.3% and 7.8% of ESCC and normal esophagus tissues (P < 0.05), respectively. SATB1 expression did not significantly correlate with clinicopathological features. The Kaplan–Meier curve indicated that patients with high SATB1 expression had significantly shorter survival than those with low SATB1 expression. In a multivariate Cox regression model, high SATB1 expression was identified as an independent prognostic factor for patients with ESCC. In conclusion, these results suggest that high SATB1 expression is predictive of poor prognosis in ESCC and may be a promising new candidate for targeted therapies for ESCC.     

NK cell dysfunction with down‐regulated CD16 and up‐regulated CD56 molecules in patients with esophageal squamous cell carcinoma

NK cells can be divided into two subsets, CD56^dim and CD56^bright NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56^dim or CD56^bright NK cells gated on CD56(+)CD3(–) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab‐mediated antibody‐dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)‐expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56^dim and CD56^bright NK cells between ESCC patients and healthy donors, down‐regulated CD16 and up‐regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab‐mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down‐regulated CD16 and up‐regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF‐beta1 partially contributed to down‐regulation of CD16 on NK cells. Down‐regulated CD16 and up‐regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.     

Upregulated ataxia‐telangiectasia group D complementing gene correlates with poor prognosis in patients with esophageal squamous cell carcinoma

The ataxia‐telangiectasia group D complementing gene (ATDC) plays significant roles in various human cancers. However, the clinical significance of ATDC in esophageal squamous cell carcinoma (ESCC) has not been investigated. The ATDC messenger RNA level of 40 paired ESCC and nonneoplastic tissues were evaluated using quantitative real‐time polymerase chain reaction, 10 pairs of which were also used for Western blot analysis. In addition, immunohistochemical staining was performed to detect the ATDC expression in 118 paraffin‐embedded cancerous and matched nonneoplastic tissues, and the correlation of ATDC expression with the clinicopathological parameters and prognosis of the ESCC patients was analyzed. The quantitative real‐time polymerase chain reaction, immunohistochemical staining, and Western blot results demonstrated that the expression level of ATDC was significantly higher in ESCC tissue than in matched noncancerous tissues. Both ATDC messenger RNA and protein expression in the ESCC tissue were significantly correlated with tumor differentiation, stage, and lymph node metastasis. However, there was no significant difference in ATDC expression based on patient age or gender. Moreover, the results of both univariate and multivariate analyses showed that increased ATDC expression was correlated with a shorter 5‐year survival time for ESCC patients after surgery. We concluded that increased ATDC expression is associated with poor clinical outcomes and that this marker might be a useful indicator for prognosis and a promising target for therapy in ESCC patients.