[Retracted] Filamin A regulates EGFR/ERK/Akt signaling and affects colorectal cancer cell growth and migration.

Following the publication of this article, an interested reader drew to the authors' attention that various panels in the scratch-wound assays shown in Fig. 1C appeared to contain overlapping sections, such that the data may have been derived from a more limited selection of original sources where the data were intended to show the data from discrete experiments. Furthermore, the results shown in the Kaplan-Meier overall survival analysis plots in Fig. 4C appeared to be inconsistent with the date of publication of this article. Independently, the Editorial Office also investigated the issues of concern in this article, and although the authors attenpted to explain these issues and requested the publication of a corrigendum, the Editor of Molecular Medicine Reports has declined this request and determined that this article should be retracted from the journal on the basis of an overall lack of confidence in the presented data. Upon receiving this decision from the Editor, the authors were not in agreement that the article should be retracted. The Editor apologizes to the readership of the Journal for any inconvenience caused. [Molecular Medicine Reports 20: 3671-3678, 2019; DOI: 10.3892/mmr.2019.10622].

Filamin A regulates EGFR/ERK/Akt signaling and affects colorectal cancer cell growth and migration.

The metastasis and recurrence rate, and the overall prognosis of colorectal cancer (CRC) remain unsatisfactory. Filamin A (FLNa), as an actin­binding protein, can interact with various signaling molecules and membrane receptors to affect cell signal transduction and function. However, whether FLNa is involved in the progression of CRC remains to be elucidated. The aim of the present study was to explore the role of FLNa in CRC cell proliferation and migration, as well as in the regulation of epidermal growth factor receptor (EGFR) signaling. Following transfection with a FLNa­targeting short hairpin RNA plasmid to knockdown expression of FLNa in the EGF­treated SW480 cell line, it was found that decreased expression of FLNa promoted cell proliferation and migration. Additionally, there was a negative correlation between FLNa levels and the activation of EGFR and Akt signaling pathways. Similarly, the expression of FLNa was significantly lower in human CRC tissues compared with adjacent normal tissues and FLNa expression was negatively correlated with the expression of Ki­67 in human CRC tissues. Although there was no significant difference in the Kaplan­Meier estimate of CRC between high expression and low expression of FLNa, there were significant negative associations between FLNa expression and TNM stage. The results suggested that FLNa may participate in EGF­induced cell proliferation and migration in CRC cells. Hence, interventions in the FLNa­mediated signaling pathway could provide attractive therapeutic targets for CRC.

Aberrant CpG Island Shore Region Methylation of CAV1 Is Associated with Tumor Progression and Poor Prognosis in Gastric Cardia Adenocarcinoma.

BACKGROUND AND AIMS: Caveolin-1 (CAV1) is a multifunctional scaffolding protein and plays an important role in tumorigenesis. However, the epigenetic changes of CAV1 in gastric cardia adenocarcinoma (GCA) have not been investigated so far. The purpose of this study was to clarify the contribution of critical CpG sites in CAV1 to progression/prognosis of GCA and to further elucidate the effect of critical CpG sites on the ectopic expression of ß-catenin in GCA. METHODS: Methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods were, respectively, applied to examine the methylation status of CAV1. RT-PCR and immunohistochemistry methods were used to determine the mRNA and protein expression of CAV1 and ß-catenin. RESULTS: Decreased mRNA and protein expression of CAV1 were observed in GCA tumor tissues and were associated with hypermethylation of CpG island shore and transcription start site (TSS) regions in CAV1. Hypermethylation of the other two regions within CpG islands in CAV1 was observed both in tumor and corresponding adjacent tissues but was not related to the transcriptional inhibition of CAV1. The methylation status of CpG island shore region in CAV1 was associated with the ectopic expression of ß-catenin and was independently associated with survival in GCA patients. CONCLUSIONS: Hypermethylation of CpG island shore and TSS regions is cancer specific and is closely associated with reduced expression of CAV1. The CpG island shore methylation of CAV1 may play an important role in progression of GCA and may serve as a prognostic methylation biomarker for GCA patients.

Expression of caveolin-1 is correlated with lung adenocarcinoma proliferation, migration, and invasion.

Both tumor suppressor and tumor promoter roles, which are dependent on the tumor type, have been described for caveolin-1 (CAV-1). Because CAV-1 can modulate cell signaling, we tested the hypothesis that it regulates lung adenocarcinoma cell proliferation and metastasis via modulation of epidermal growth factor receptor (EGFR) activity. The lung adenocarcinoma cell line, GLC-82, was transfected with pcDNA3.1CAV-1 plasmid, before cell proliferation, migration, and invasion were analyzed. In the in vivo xenograft model, the relationship between the CAV-1 expression and EGFR phosphorylation and signaling was assessed by western blot analysis. The relationship between the CAV-1 as well as Ki67 expression and the clinicopathological characteristics of 68 lung adenocarcinoma patients was also examined using immunohistochemistry. Overexpression of CAV-1 significantly increased GLC-82 proliferation (p < 0.001), migration (p < 0.001), and invasion (p = 0.002) as well as EGFR and ERK phosphorylation (p < 0.05). The GLC-82/CAV-1 cell tumors were also significantly larger than those of control cells (all p ≤ 0.05). In lung adenocarcinoma patients, CAV-1 expression was positively correlated with lymph node metastasis and cancer stage. Finally, CAV-1 expression was associated with the expression of Ki-67, a marker of cell proliferation. CAV-1 enhanced GLC-82 cell proliferation, migration, and invasion possibly through EGFR and ERK signaling. Furthermore, the relationship of CAV-1 with Ki67 expression, a marker of proliferative capacity, in lung adenocarcinoma samples is suggestive of its role in disease progression. Further studies are required to confirm the role of CAV-1 in the metastasis of lung adenocarcinoma as well as its potential prognostic and therapeutic value.

Filamin A modulates kinase activation and intracellular trafficking of epidermal growth factor receptors in human melanoma cells.

The actin-binding protein filamin A (FLNa) affects the intracellular trafficking of various classes of receptors and has a potential role in oncogenesis. However, it is unclear whether FLNa regulates the signaling capacity and/or down-regulation of the activated epidermal growth factor receptor (EGFR). Here it is shown that partial knockdown of FLNa gene expression blocked ligand-induced EGFR responses in metastatic human melanomas. To gain greater insights into the role of FLNa in EGFR activation and intracellular sorting, we used M2 melanoma cells that lack endogenous FLNa and a subclone in which human FLNa cDNA has been stably reintroduced (M2A7 cells). Both tyrosine phosphorylation and ubiquitination of EGFR were significantly lower in epidermal growth factor (EGF)-stimulated M2 cells when compared with M2A7 cells. Moreover, the lack of FLNa interfered with EGFR interaction with the ubiquitin ligase c-Cbl. M2 cells exhibited marked resistance to EGF-induced receptor degradation, which was very active in M2A7 cells. Despite comparable rates of EGF-mediated receptor endocytosis, internalized EGFR colocalized with the lysosomal marker lysosome-associated membrane protein-1 in M2A7 cells but not M2 cells, in which EGFR was found to be sequestered in large vesicles and subsequently accumulated in punctated perinuclear structures after EGF stimulation. These results suggest the requirement of FLNa for efficient EGFR kinase activation and the sorting of endocytosed receptors into the degradation pathway.

NS-398 induces apoptosis in human esophageal cancer cells through inhibition of NF-kappaB downstream regulation of cyclooxygenase-2.

Although non-steroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to have cancer-preventive effects and induce apoptosis of cancer cells, the mechanism of their effects is not clearly known. We studied the mechanism in human esophageal cancer cell line TE13. The esophageal squamous cell carcinoma cell line TE-13 was cultured with NS-398 at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Prostaglandin F(1alpha) was determined with radioimmunoassay. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. NS-398 significantly inhibited cell proliferation and induced apoptosis at concentrations of 0.001, 0.01, 1, and 100 micromol/L. NS-398 dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein, nuclear NF-kappaB protein and production of PGF(1alpha) and increased the cytoplasmic IkappaB protein. In conclusion, NS-398 inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB.

The effects of a COX-2 inhibitor meloxicam on squamous cell carcinoma of the esophagus in vivo.

Our previous study showed that aspirin induced apoptosis of esophageal cancer cells in vitro by inhibiting the pathway of NF-kappaB downstream regulation of cyclooxygenase-2. The purpose of this study was to determine if similar changes occurred in vivo in the tumors of patients with SCC of the esophagus who were given a preferential COX-2 inhibitor, meloxicam. Fifty-three patients who had an esophagectomy for SCC were allocated randomly to either a Treatment group (n = 25) or a control group (n = 28). Patients in the Treatment group were given 7.5 mg/day of meloxicam, for between 10 and 14 days before surgery. Patients in the control group did not take any type of NSAID during this time interval. Samples of the tumor taken from the resected specimens were collected. Proliferation and apoptosis were measured by flow cytometry. The concentration of 6-keto-prostaglandin F(1)alpha in cancer tissue was determined by radio-immuno-assay. Expression of COX-2 mRNA was measured with RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic I kappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. There were significantly more apoptotic cells in the tumors of patients who were using meloxicam. It also decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic I kappaB protein in the cancer. We conclude that meloxicam induces apoptosis in SCC of the esophagus in vivo by inhibiting the pathway of NF-kappaB downstream regulation of COX-2.

Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor kappaB.

The DNA binding protein nuclear factor kappaB (NF-kappaB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-kappaB also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappaB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-kappaB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-kappaB consensus sequence. The activated NF-kappaB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials--(i) purified recombinant NF- kappaB p50, (ii) nuclear extracts, and (iii) whole cell lysates--we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-kappaB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappaB activation.

Filamin A-mediated down-regulation of the exchange factor Ras-GRF1 correlates with decreased matrix metalloproteinase-9 expression in human melanoma cells.

The actin-binding protein filamin A (FLNa) is associated with diverse cellular processes such as cell motility and signaling through its scaffolding properties. Here we examine the effect of FLNa on the regulation of signaling pathways that control the expression of matrix metalloproteinases (MMPs). The lack of FLNa in human M2 melanoma cells was associated with constitutive and phorbol ester-induced expression and secretion of active MMP-9 in the absence of MMP-2 up-regulation. M2 cells displayed stronger MMP-9 production and activity than their M2A7 counterparts where FLNa had been stably reintroduced. Using an MMP-9 promoter construct (pMMP-9-Luc), in vitro kinase assays, and genetic and pharmacological approaches, we demonstrate that FLNa mediated transcriptional down-regulation of pMMP-9-Luc by suppressing the constitutive hyperactivity of the Ras/MAPK extracellular signal-regulated kinase (ERK) cascade. Experimental evidence indicated that this phenomenon was associated with destabilization and ubiquitylation of Ras-GRF1, a guanine nucleotide exchange factor that activates H-Ras by facilitating the release of GDP. Ectopic expression of Ras-GRF1 was accompanied by ERK activation and elevated levels of MMP-9 in M2A7 cells, whereas a catalytically inactive dominant negative Ras-GRF1, which prevented ERK activation, reduced MMP-9 expression in M2 cells. Our results indicate that expression of FLNa regulates constitutive activation of the Ras/ERK pathway partly through a Ras-GRF1 mechanism to modulate the production of MMP-9.

Pyrrolidine dithiocarbamate inhibits interleukin-6 signaling through impaired STAT3 activation and association with transcriptional coactivators in hepatocytes.

Interleukin (IL)-6 is a proinflammatory cytokine that has been implicated in the expression of acute phase plasma proteins and hepatic insulin resistance through activation of the JAK/STAT3 pathway. Although previous studies have demonstrated that pyrrolidine dithiocarbamate (PDTC) exerts protection against inflammatory responses, its role in the regulation of IL-6 receptor signaling remains unclear. Here we show that treatment of cultured HepG2 hepatoma cells with PDTC inhibits IL-6-stimulated tyrosine phosphorylation and subsequent nuclear translocation of STAT3 in a dose- and time-dependent fashion. No inhibition of JAK-1 activity was observed. To provide insight into PDTC signaling, we constructed a conditionally active STAT3 by fusing it with the ligand binding domain of the estrogen receptor (STAT3-ER). In the presence of 4-hydroxytamoxifen STAT3-ER was translocated in the nucleus of HepG2 cells in a phosphorylation-independent manner, and treatment with PDTC mitigated the response. Although STAT3 coprecipitated with heat-shock protein 90 (Hsp90) in control cells, coprecipitation of the two proteins was greatly reduced after PDTC treatment or after exposure to geldanamycin, an Hsp90 inhibitor. As a result there was a decrease in IL-6-induced association of STAT3 with the transcriptional coactivators FOXO1a and C/EBPbeta together with significant reduction in the expression of SOCS-3 protein and that of two major acute phase plasma proteins. Importantly, treatment of HepG2 cells and a primary culture of rat hepatocytes with PDTC restored insulin responsiveness that was abrogated by IL-6. These studies are consistent with the ability of PDTC to down-regulate IL-6-induced STAT3 activation by altering the stability of STAT3-Hsp90 complex.

Binding of manumycin A inhibits IkappaB kinase beta activity.

IkappaB kinase (IKK) catalytic subunits play a key role in cytokinemediated nuclear factor (NF)-kappaB signaling, and a loss of NF-kappaB function appears to inhibit inflammation and oncogenesis. Manumycin A is a potent and selective farnesyltransferase inhibitor with antitumor activity. We found that manumycin A caused a rapid and potent inhibition of IKK activity induced by tumor necrosis factor alpha in a number of cell types. Most unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, cultured human HepG2 hepatoma cells were transiently transfected with various IKKalpha and IKKbeta constructs, and a striking difference in manumycin A sensitivity was observed. Furthermore, cells expressing wild-type IKKbeta and IKKbeta mutated in the activation loop at Cys-179 exhibited covalent homotypic dimerization of IKKbeta in response to manumycin A, whereas substitution of Cys-662 and -716 conferred protection against dimer formation. Direct inhibition of IKK activity and formation of stable IKKbeta dimers were observed in the presence of manumycin A that could be blocked by dithiothreitol. IKK interaction with the adaptor protein IKKgamma/NEMO was disrupted in manumycin A-treated cells. Most importantly, administration of manumycin A to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects. Thus, manumycin A with its epoxyquinoid moieties plays an important regulatory function in IKK signaling through pathways distinct from its role as a protein farnesylation inhibitor.

Aspirin induces apoptosis in oesophageal cancer cells by inhibiting the pathway of NF-kappaB downstream regulation of cyclooxygenase-2.

BACKGROUND: Aspirin has potential in the prevention or treatment of oesophageal cancer, the seventh most common cancer in the world, but its mechanism of action is still not certain. METHODS: The oesophageal squamous cell carcinoma cell line TE-13 was cultured with aspirin at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. RESULTS: Aspirin significantly inhibited cell proliferation and induced apoptosis at concentrations of 1, 4, 8 mmol/L. Aspirin dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic IkappaB protein. CONCLUSION: We conclude that aspirin inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB. These results support the further investigation of the cyclooxygenase pathway in investigating the potential of aspirin and similar drugs in cancer prevention and therapy.

Multiple factors contributing to lipopolysaccharide-induced reactivity changes in rabbit pulmonary artery.

To explore the underlying mechanism(s) of pulmonary arterial hypertension in endotoxic shock, the roles of N-acetylcysteine (NAC), nitric oxide (NO) and carbon monoxide (CO) were investigated. Pulmonary arterial rings (3-mm width) were prepared from 24 rabbits. Lipopolysaccharide (LPS), after 7-hour incubation, decreased the endothelium-dependent relaxation response of the arterial ring (pre-contracted with phenylephrine) to acetylcholine (1 mumol/L), but did not affect the endothelium-independent relaxation response to sodium nitroprusside. The LPS effects were reduced by a concomitant incubation with the free radical scavenger (NAC), NO donor (L-arginine), and CO donor (hemin), respectively. On the other hand, the LPS effects were enhanced by applying heme oxygenase-1 (HO-1) inhibitor (zinc protoporphyrin) to block CO production. The response to acetylcholine changed from relaxation to contraction, however, the contractile response to phenylephrine increased significantly after pre-incubation with nitric oxide synthase (NOS) inhibitor (L-NAME) to block NO production, confirming the importance of CO and NO. These results show that LPS impairs endothelium-dependent relaxation of the pulmonary artery, which can be greatly reduced by the antioxidant, or by supplying with NO and CO. Thus, multiple factors are involved in this model of endotoxin-induced pulmonary hypertension.

[Endogenous peroxynitrite mediates lipopolysaccharide-induced injury in cultured pulmonary artery endothelial cells].

This study, using cultured bovine pulmonary artery endothelial cells (BPAECs), was undertaken to investigate the roles of endogenous ONOO(-) in LPS-caused injury in endothelial cells. The fluorescent intensity of nitrotyrosine (NT), a specific marker of ONOO(-) generation, in BPAECs represented the content of endogenous ONOO(-) generation. The fluorescent intensity of NT and the number of NT positive cells were detected with flow cytometry (FCM), and the percentage of NT positive cells was calculated. The results are as follows. (1) LPS (1, 5 and 10 microg/ml) caused a marked increase in fluorescent intensity of NT in a dose-dependent manner, which was significantly increased compared to the vehicle group (P<0.01).The number and percentage of NT positive cells were markedly increased (both P<0.05 vs vehicle group). Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase (iNOS), inhibited LPS-induced increase in fluorescent intensity of NT in BPAECs. However, the number and percentage of NT positive cells had a tendency to reduce. (2) LPS brought about an enhancement in MDA content and the activity of LDH in cultured supernatant. AG reversed the enhancement in MDA content induced by LPS (P<0.01). In contrast, AG had a marginal effect on the activity of LDH. (3) LPS induced an increase in apoptotic rate in BPAECs in a dose-dependent manner. The number of apoptotic cells markedly increased as well. Some BPAECs stained with fluorescent probe ethidium bromide showed morphological features of apoptosis with chromatin condensation and nuclear fragmentation. AG reduced the apoptotic rate and the number of apoptotic cells, both of which were still higher than those of vehicle group (P<0.05). LPS led to inhibition of mitochondrial respiration and membrane potential in an accumulation manner. In conclusion, LPS caused injury to cultured BPAECs and increased the production of ONOO(-).The cytotoxicity of LPS may be mediated by the endogenous ONOO(-).

[The role of N-acetylcysteine against the injury of pulmonary artery induced by LPS].

AIM: To investigate the alleviating effect of N-acetylcysteine (NAC) on lung injury induced by lipopolysaccharides (LPS) and its mechanism. METHODS: The effects of NAC on changes of the pulmonary arterial reactivity and the ultrastructure of pulmonary arterial endothelium induced by LPS were observed with the isolated artery ring technique and scanning electron microscope (SEM). Malondialdehyde (MDA), nitric oxide (NO) contents and superoxide dismutase (SOD) activity of pulmonary artery tissues were detected. RESULTS: The exposure of pulmonary artery to LPS (4 microg/ml, 7 h) led to reduction of endothelium-dependent relaxation response to acetylcholine (ACh), which was reversed by the concomitant exposure to NAC (0.5 mmol/L, 7 h), whereas NAC itself had no effect on the response. Significant structural injury were observed under SEM in LPS group and alleviated the changes in LPS + NAC group. The MDA, NO contents increased but SOD activity decreased in LPS group, which were reversed by the concomitant exposure to NAC. CONCLUSION: NAC protects pulmonary artery endothelium and enhances endothelium-dependent relaxation response of pulmonary artery by antioxidation effect, which may be one of the mechanisms of its reversing pulmonary artery hypertension and following lung injury induced by LPS.