Role of Integrin ß1 in the progression and chemo-resistance of esophageal squamous cell carcinoma.

Objective: Integrins have been shown to play an important role in the tumorigenesis of many cancers. In this work, we aimed to explore the expression and clinical value of Integrin α5ß1 in esophageal squamous cell carcinoma (ESCC), and the effect of integrin ß1 on the development and chemo-resistance of ESCC cells. Methods: The expression profiling of integrins was analyzed in the mRNA expression dataset of ESCC. The expression of Integrin α5ß1 in 278 cases of ESCC tissues and 62 cases of paracancerous tissues was detected by immunohistochemistry (IHC). The association between the expression of Integrin α5ß1 and the survival of ESCC patients was analyzed by Kaplan-Meier analysis. The effect of Integrin ß1 on the proliferation, migration, and invasion of ESCC cells was examined by MTS, Transwell migration, and Transwell invasion assay. The effect of Integrin ß1 and L1 cell adhesion molecule (L1CAM) on cisplatin resistance was detected by MTS and the signal pathways involved were analyzed by Western blotting. Results: Integrin ß1 and Integrin α5 were significantly up-regulated in ESCC. High expression of Integrin ß1 was also related to worse overall survival of ESCC patients and patients with low levels of both Integrin ß1 and Integrin α5 showed the shortest survival. Results of IHC revealed that Integrin α5ß1 was up-regulated in ESCC and its high expression was associated with poor prognosis and could serve as an independent prognostic factor. siRNA-mediated Integrin ß1 silencing or antibody blocking restrained the proliferation, migration, and invasion of ESCC cells. Simultaneous knockdown of Integrin ß1 and L1CAM reduced the cisplatin resistance of ESCC cells. Further studies showed that knockdown of Integrin ß1 and L1CAM suppressed the activity of Akt signaling with or without cisplatin treatment. Moreover, dual high expression of Integrin ß1 and L1CAM was related to worse overall survival of ESCC patients treated with preoperative chemotherapy. Conclusion: Integrin α5ß1 was up-regulated in ESCC and could be used as a new prognostic indicator for ESCC patients. In addition, Integrin ß1 was involved in the proliferation, invasion, and chemo-resistance of ESCC cells.

Promotion of rs3746804 (p. L267P) polymorphism to intracellular SLC52A3a trafficking and riboflavin transportation in esophageal cancer cells.

Riboflavin is an essential micronutrient for normal cellular growth and function. Lack of dietary riboflavin is associated with an increased risk for esophageal squamous cell carcinoma (ESCC). Previous studies have identified that the human riboflavin transporter SLC52A3a isoform (encoded by SLC52A3) plays a prominent role in esophageal cancer cell riboflavin transportation. Furthermore, SLC52A3 gene single nucleotide polymorphisms rs3746804 (T>C, L267P) and rs3746803 (C >T, T278M) are associated with ESCC risk. However, whether SLC52A3a (p.L267P) and (p.T278M) act in riboflavin transportation in esophageal cancer cell remains inconclusive. Here, we constructed the full-length SLC52A3a protein fused to green fluorescent protein (GFP-SLC52A3a-WT and mutants L267P, T278M, and L267P/T278M). It was confirmed by immunofluorescence-based confocal microscopy that SLC52A3a-WT, L267P, T278M, and L267P/T278M expressed in cell membrane, as well as in a variety of intracellular punctate structures. The live cell confocal imaging showed that SLC52A3a-L267P and L267P/T278M increased the intracellular trafficking of SLC52A3a in ESCC cells. Fluorescence recovery after photobleaching of GFP-tagged SLC52A3a meant that intracellular trafficking of SLC52A3a-L267P and L267P/T278M was rapid dynamics process, leading to its stronger ability to transport riboflavin. Taken together, the above results indicated that the rs3746804 (p.L267P) polymorphism promoted intracellular trafficking of SLC52A3a and riboflavin transportation in ESCC cells.

Heat shock protein family B member 1 facilitates ezrin activation to control cell migration in esophageal squamous cell carcinoma.

Ezrin plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC), providing a link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and protrusions. However, the mechanism by which ezrin is activated still remains unknown in ESCC. Here, we identify a novel interaction between ezrin and heat shock protein family B (small) member 1 (HSPB1) in ESCC cells by mass spectroscopy and co-immunoprecipitation. HSPB1 only interacts with inactive ezrin and binds to the α-helical coiled coil region of ezrin. Knockdown of HSPB1 resulted to the decline of phosphorylation at ezrin Thr567, markedly suppressing the ability of ezrin to bind to the actin cytoskeleton and migration of ESCC cells. Furthermore, neither the constitutively active phosphomimetic ezrin T567D, nor inactivated ezrin T567A could restore cell migration following HSPB1 knockdown. Low HSPB1 expression was associated with favorable overall survival of ESCC patients. Taken together, HSPB1, as an important partner, participates in the activation of ezrin and merits further evaluation as a novel therapeutic target against human ESCC.

F806 Suppresses the Invasion and Metastasis of Esophageal Squamous Cell Carcinoma via Downregulating F-Actin Assembly-Related Rho Family Proteins.

Invasion and metastasis are critical pathological and mortal processes in esophageal squamous cell carcinoma (ESCC). Novel drugs, targeting the two cancer migration stages, will augment the treatment options for ESCC therapy and improve overall survival. A novel natural macrolide F806 specifically promotes apoptosis of various ESCC cells. However, whether F806 can inhibit metastasis of ESCC cells needs further evaluation. Here, our data showed that F806 inhibits dynamic F-actin assembly and then suppresses the migration of ESCC cells in vitro and their invasion and metastasis in vivo. The correlation between cancer migration and actin cytoskeleton assembly was consistent with the ability of F806 to prevent the aggregation of Paxillin, an essential protein for focal adhesion formation through binding to the ends of actin filaments. Furthermore, F806 downregulated the expression and activity of the Rho family proteins cell division cycle 42 (CDC42), RAC family small GTPase 1 (RAC1), and RAS homolog family member A (RHOA). Taken together, these results suggest that F806 can suppress cancer invasion and metastasis via interrupting the assembly of migration components involving F-actin.

The interaction of lncRNA EZR-AS1 with SMYD3 maintains overexpression of EZR in ESCC cells.

EZR, a member of the ezrin-radixin-moesin (ERM) family, is involved in multiple aspects of cell migration and cancer. SMYD3, a histone H3-lysine 4 (H3-K4)-specific methyltransferase, regulates EZR gene transcription, but the molecular mechanisms of epigenetic regulation remain ill-defined. Here, we show that antisense lncRNA EZR-AS1 was positively correlated with EZR expression in both human esophageal squamous cell carcinoma (ESCC) tissues and cell lines. Both in vivo and in vitro studies revealed that EZR-AS1 promoted cell migration through up-regulation of EZR expression. Mechanistically, antisense lncRNA EZR-AS1 formed a complex with RNA polymerase II to activate the transcription of EZR. Moreover, EZR-AS1 could recruit SMYD3 to a binding site, present in a GC-rich region downstream of the EZR promoter, causing the binding of SMYD3 and local enrichment of H3K4me3. Finally, the interaction of EZR-AS1 with SMYD3 further enhanced EZR transcription and expression. Our findings suggest that antisense lncRNA EZR-AS1, as a member of an RNA polymerase complex and through enhanced SMYD3-dependent H3K4 methylation, plays an important role in enhancing transcription of the EZR gene to promote the mobility and invasiveness of human cancer cells.

Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation.

BACKGROUND: Ezrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear. METHODS: Distribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis. RESULTS: Ezrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo. CONCLUSION: Ezrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.

Hydrogen sulfide reduces regional myocardial ischemia injury through protection of mitochondrial function.

Hydrogen sulfide (H2S) is a signaling gasotransmitter, involved in various physiological and pathological processes. H2S-donating drugs have been tested to conjugate the beneficial effects of H2S with other pharmaceutical properties. It has been shown that the endogenous cystathionine-γ-lyase (CSE)/H2S pathway participates in myocardial ischemia injury in isolated hearts in rats. The present study aimed to investigate the cytoprotective action of H2S against acute myocardial ischemia injury in rats. Isolated rat hearts were perfused and subjected to ischemic conditions for 4 h. The hearts were assigned to five groups: Sham, model, infarct plus low-dose (5 µmol/l) NaHS, infarct plus middle-dose (10 µmol/l) NaHS and infarct plus high-dose (20 µmol/l) NaHS. The administration of NaHS enhanced the activity of CSE, increased the content of H2S and reduced infarct volumes following myocardial ischemia injury. Furthermore, the administration of NaHS attenuated the injury to organelles (including the mitochondria, nucleus and myofilaments) by reducing lactate dehydrogenase activity, decreasing the level of mitochondrial malondialdehyde and increasing the activities of superoxide dismutase and glutathione peroxidase in the ischemic myocardial mitochondria. These protective effects of H2S against myocardial ischemia injury appeared to be mediated by its antioxidant activities and the preservation of mitochondrial function.

Crosstalk between dopamine receptors and the Na⁺/K⁺-ATPase (review).

Dopamine (DA) receptors, which belong to the G protein-coupled receptor family, are the target of ~50% of all modern medicinal drugs and constitute a large and diverse class of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. Na+/K+-ATPase (NKA) is ubiquitous and crucial for the maintenance of intracellular ion homeostasis and excitability. Furthermore, it plays a critical role in diverse effects, including clinical cardiotonic and cardioprotective effects, ischemic preconditioning in the brain, natriuresis, lung edema clearance and other processes. NKA regulation is of physiological and pharmacological importance and has species- and tissue-specific variations. The activation of DA receptors regulates NKA expression/activity and trafficking in various tissues and cells, for example in the kidney, lung, intestine, brain, non-pigmented ciliary epithelium and the vascular bed. DA receptor-mediated regulation of NKA mediates a diverse range of cellular responses and includes endocytosis/exocytosis, phosphorylation/dephosphorylation of the α subunit of NKA and multiple signaling pathways, including phosphatidylinositol (PI)-phospholipase C/protein kinase (PK) C, cAMP/PKA, PI3K, adaptor protein 2, tyrosine phosphatase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase. Furthermore, in brain and HEK293T cells, D1 and D2 receptors exist in a complex with NKA. Among D1 and D2 receptors and NKA, regulations are reciprocal, which leads to crosstalk between DA receptors and NKA. In the present study, the current understanding of signaling mechanisms responsible for the crosstalk between DA receptors and NKA, as well as with specific consequent functions, is reviewed.