The inhibition of Beclin1-dependent autophagy sensitizes PTC cells to ABT737-induced death.

ABT737 is used as a specific BCL2 inhibitor, which can treat papillary thyroid carcinoma (PTC). However, the effect of ABT737 on PTC cell apoptosis is limited. Moreover, BCL2 inhibition causes the activation of Beclin1-dependent autophagy. Our study aimed to explore the effects of autophagy and Beclin1 on ABT737 efficacy in PTC. The experimental data showed that ABT737 synchronously enhanced autophagic activity and apoptosis level in PTC cells. ABT737 also promoted the dissociation of BCL2-Beclin1 and BCL2-Bax complexes. Autophagy inhibitors, Bafilomycin A1 and 3-MA, enhanced the inhibitory effect of ABT737 on the survival and function in PTC cells. Consistently, autophagy inhibition with Beclin1 pharmacological inhibitor (spautin-1) also enhanced the efficacy of ABT737. Additionally, ABT737 at low-dose promoted LC3 conversion in PTC cells, and did not affect PTC cell apoptosis and survival. However, The efficacy of low-dose of ABT737 in PTC cell apoptosis and survival was displayed with the addition of Bafilomycin A1, 3-MA or spautin-1. In conclusion, the limited role of ABT737 in PTC cell apoptosis is attributed to its promoting effect on Beclin1-dependent autophagy. Therefore, autophagy inhibition based on Beclin1 downregulation can enhance the sensitivity of PTC cells to ABT737-induced death.

PHACTR1 promotes the mobility of papillary thyroid carcinoma cells by inducing F-actin formation.

Papillary thyroid carcinoma (PTC) limits effective biomarkers for predicting prognosis and targeted therapy. Phosphatase and actin regulator 1 (PHACTR1) is a mobility-promoting molecule due to its regulation on F-actin formation, which is valuable for the investigation of PTC. Our study aimed to investigate the relationship between PHACTR1 and PTC carcinogenesis, especially mobility. Our results displayed that PHACTR1 expression was elevated in metastatic or larger PTC tissues. In addition, PTC cells K1 with more obvious mobility had higher PHACTR1 expression whereas weakly mobile cells TPC-1 was contrary. Moreover, PHACTR1 silencing inhibited the invasion, migration and tumorigenicity of K1 cells, while PHACTR1 overexpression promoted the invasion, migration and tumorigenicity in TPC-1 cells. Furthermore, PHACTR1 overexpression increased the fluorescent intensity of F-actin in TPC-1 cells. Importantly, the enhanced invasion and migration in TPC-1 cells caused by PHACTR1 overexpression were significantly reversed by the disruption of F-actin assembly with swinholide A. In conclusion, PHACTR1 can promote the mobility of PTC cells, which results in the carcinogenesis of PTC. PHACTR1-regulated F-actin formation determines the mobility of PTC cells. Therefore, PHACTR1 can function as a potential biomarker for predicting prognosis and targeting therapy in PTC.

Electrospun membranes of carboxylated poly(ester urethane)urea/gelatin encapsulating pterostilbene for adaptive and antioxidative purposes.

Oxidative stress caused by the harsh microenvironment after implantation of an artificial graft with mismatching mechanical properties usually triggers inflammation responses, which have adverse impacts on tissue regeneration. For coping with these problems, in this work, bioactive fibrous scaffolds were developed from specially synthesized carboxylated poly(ester urethane)urea (PEUU) and gelatin (Gel) by encapsulating pterostilbene (Pte) for the first time. The prepared electrospun membranes exhibited self-adaptable mechanical properties with high elasticity owing to the bonded electrospun fibers, cross-linking network between PEUU and Gel, and the inherent flexibility of the PEUU polymer in the fibrous matrix. The PEUU/Gel/Pte electrospun membrane containing 7% Pte could promote in vitro proliferation of human umbilical vein endothelial cells, and regulate vascular smooth muscle cells with excellent antioxidant properties via free radical scavenging. In vivo results in a rat subcutaneous implantation model further demonstrated the positive effect of the specially prepared PEUU/Gel/Pte scaffold on both normal cell growth and anti-inflammatory by promoting cellularization and polarizing macrophages toward the M2 phenotype. These PEUU/Gel/Pte electrospun membranes with adaptability benefit to tissue regeneration by modulating inflammation responses, especially applications in vascular regeneration.

TAT-Beclin 1 represses the carcinogenesis of DUSP4-positive PTC by enhancing autophagy.

BACKGROUND: DUSP4 is a pro-tumorigenic molecule of papillary thyroid carcinoma (PTC). DUSP4 also exists as an autophagic regulator. Moreover, DUSP4, as a negative regulator of MAPK, can prevent Beclin 1 from participating in autophagic response. This study aimed to explore whether TAT-Beclin 1, a recombinant protein of Beclin 1, could inhibit the tumorigenesis of DUSP4-positive PTC by regulating autophagy. METHODS: First, we divided PTC tissues into three groups according to DUSP4 expression levels by immunohistochemical analyses, and evaluated the relationship between autophagic molecules (Beclin 1 and LC3II) and DUSP4 using Western blotting assays. After overexpression of DUSP4 by lentiviral transduction, the in vitro and in vivo roles of TAT-Beclin 1 on DUSP4-overexpressed PTC cells were assessed (including autophagic activity, cell survival and function, and tumor growth). The roles of TAT-Beclin 1 in the survival of DUSP4-silenced PTC cells were also evaluated. RESULTS: Our results showed that the expression levels of autophagic proteins decreased with the increase of DUSP4 expression in PTC tissues. In PTC cells, DUSP4 overexpression-inhibited autophagic activity (including Beclin 1 expression, LC3 conversion rate and LC3-puncta formation) and -promoted cell proliferation and migration were reversed by TAT-Beclin 1 administration. In vivo assays also showed that DUSP4-overexpressed PTC cells had stronger tumorigenic ability and weaker autophagic activity, which was blocked by TAT-Beclin 1 administration. CONCLUSION: TAT-Beclin 1, as an autophagic promoter, could repress the carcinogenesis of DUSP4-positive PTC, which implies that the use of TAT-Beclin 1 for the PTC patients' treatment might be determined according to the DUSP4 level in their tumors.

miR­122­5p suppresses the oncogenesis of PTC by inhibiting DUSP4 expression.

MicroRNAs (miRNAs or miRs) play an important role in regulating the occurrence and development of papillary thyroid carcinoma (PTC). miR­122­5p is widely considered a tumour inhibitor, which has not been fully explored in PTC. Bioinformatics analysis identified dual specificity phosphatase 4 (DUSP4), a tumour promoter gene for PTC, as a downstream target of miR­122­5p. The aim of the present study was to investigate the role and molecular mechanism of miR­122­5p in PTC oncogenesis. In this study, the expression pattern of miR­122­5p in PTC cancer tissues and PTC cell lines was investigated via reverse transcription­quantitative PCR. Furthermore, the roles of miR­122­5p in PTC were explored using gain­of­function and loss­of­function assays. The results revealed that the expression of miR­122­5p was significantly lower in PTC cancer tissues, especially in cancer tissues with significant invasion or metastasis. Overexpression of miR­122­5p caused by miR­122­5p mimics inhibited the proliferation, invasion, and migration of the PTC cell line K1, while knockdown of miR­122­5p by miR­122­5p inhibitors exhibited the opposite effect. Furthermore, in vivo assays revealed that miR­122­5p overexpression inhibited tumour growth. In addition, miR­122­5p was negatively correlated with DUSP4 expression in PTC cancer tissues. miR­122­5p overexpression inhibited DUSP4 expression in K1 cells, while miR­122­5p downregulation produced the inverse effect. Specifically, a luciferase reporter assay confirmed the binding sites of miR­122­5p on the 3'­UTR of DUSP4, demonstrating the targeting effect of miR­122­5p on DUSP4. miR­122­5p inhibited the oncogenesis of PTC by targeting DUSP4, revealing the potential application value of miR­122­5p in the diagnosis and treatment of PTC.

Hsa_circ_0011290 regulates proliferation, apoptosis and glycolytic phenotype in papillary thyroid cancer via miR-1252/ FSTL1 signal pathway.

OBJECTIVE: Despite of previous report regarding the aberrant overexpression of hsa_circ_0011290 in thyroid cancer, the regulatory mechanism and mechanistic involvements of which were still elusive currently in papillary thyroid cancer (PTC). Here we set out to characterize expression status and functional contributions of hsa_circ_0011290 in this disease especially through mode-of-action of sponging RNA. METHODS: Relative expression of hsa_circ_0011290, microRNA (miR)-1252 and FSTL1 was quantified by real-time polymerase chain reaction. Glucose metabolism was determined by examination of glucose uptake, lactate production and ATP contents. The regulatory effects of miR-1252 on both hsa_circ_0011290 and Follistatin Like 1 (FSTL1) were interrogated by luciferase reporter assay. Direct binding between miR-1252 with hsa_circ_0011290 and FSTL1 transcripts were analyzed by RNA pulldown assay. Protein levels of FSTL1 was examined by Western blots. RESULTS: Aberrant over-expression of hsa_circ_0011290 was associated with advanced stage and unfavorable prognosis of PTC. Knockdown of hsa_circ_0011290 greatly inhibited cell viability, proliferation and stimulated cell apoptosis in PTC cells. Meanwhile, glucose metabolism was significantly switched with decreased glucose uptake and lactate production, and increased ATP contents. We identified miR-1252 as target miR of hsa_circ_0011290, and miR-1252 evidently inhibited expressions of both luciferase reporter and endogenous hsa_circ_0011290, and miR-1252 was negatively regulated by hsa_circ_0011290 vice versa. We further suggested that FSTL1 as direct target of miR-1252, and provided direct evidences in support of binding between miR-1252 with both hsa_circ_0011290 and FSTL1. Through sponging miR-1252, hsa_circ_0011290 was capable of positively modulate FSTL1 expression. Notably, inhibition of miR-1252 completely reversed phenotypic effects of hsa_circ_0011290 knockdown including cell viability, proliferation, apoptosis and glucose metabolisms. CONCLUSION: Our study uncovered the oncogenic contributions of hsa_circ_0011290-miR-1252-FSTL1 in PTCs.

Quercetin alleviates hyperthyroidism-induced liver damage via Nrf2 Signaling pathway.

Quercetin is a plant flavonoid and has antioxidative properties. In this study, we evaluated the therapeutic effect of quercetin on thyroid dysfunction in L-thyroxin (LT4)-induced hyperthyroidism rats. LT4 was used to prepare the experimental hyperthyroidism model via the intraperitoneal injection. Quercetin was injected at a series doses (5, 50, and 100 mg/kg) to LT4-induced hypothyroidism rats once a day for 14 days. The body weight and food intake were measured once a week. The levels of thyroid hormones, liver function, oxidative stress markers, and antioxidant markers were measured using commercial enzyme-linked immunosorbent assay kits. Hematoxylin-eosin staining was used to observe the thyroid tissue histological changes. The levels of nuclear and total nuclear factor erythroid 2-related factor 2 (Nrf2) were determined by western blot. The liver oxidative stress markers in LT4-induced hyperthyroidism Nrf2 knockout rats were determined to evaluate the role of Nrf2 on quercetin induced protective effects. LT4 administration increased the levels of serum triiodothyronine and thyroxine, activity of oxidative stress markers with a parallel decrease in antioxidant markers and Nrf2. However, the simultaneous administration of quercetin, reversed all these effects indicating its potential in the regulation of hyperthyroidism. Furthermore, the loss function of Nrf2 diminished these effects resulting from the quercetin application, indicating the inhibitory effects caused by the quercetin may be involved in Nrf2 signaling pathway. These results indicate that quercetin could be used to protect against experimental hyperthyroidism-induced liver damage via Nrf2 signaling pathway.

[Airway epithelial cells increase macrophage chemotaxis and inflammatory cytokine secretion under hypoxic conditions].

OBJECTIVE: To investigate the effects of airway epithelial cells on macrophages chemotaxis and inflammatory cytokine expression under hypoxic conditions.
 Methods: Human bronchial epithelial cells (HBE) treated with different concentrations (0, 100, 200, 400, 800 µmol/L) of CoCl2 or transfected with HIF-1α siRNA were co-cultured with THP-1-derived M1 macrophages or M2 macrophages. The chemotactic effects on macrophages were analyzed by Transwell assay. The levels of TNF-α, IFN-γ, IL-4, IL-13 and IL-10 in the supernatants of macrophages were detected by ELISA, and HIF-1α or Cav-1 mRNA expression in HBE or macrophages was detected by RT-qPCR.
 Results: HBE cells promoted macrophages chemotaxis in a time- and concentration-dependent manner. Compared to un-transfected group, the chemotactic ability of HBE transfected with HIF-1α siRNA was significantly weakened (P<0.01). Under the same culture conditions, the chemotaxis of M2 macrophages was greater than that in THP1-derived M1 macrophages. The concentrations of TNF-α, IFN-γ, IL-4, IL-13 and IL-10 in the supernatants of macrophages were increased in a time-and concentration-dependent manner. The concentrations of TNF-α and IFN-γ were increased further after co-culturing for 8 and 12 h; while IL-4, IL-13 and IL-10 concentrations were increased further during 24 h of co-culture. The levels of cytokines in the supernatants of macrophages co-cultured with HBE and transfected with HIF-1α siRNA were significantly lower than those in un-transfected cells (P<0.05 or P<0.01). The reduction of TNF-α or IFN-γ was more obvious. The expression of HIF-1α or Cav-1 mRNA in HBE or macrophages was increased in a concentration-dependent manner after 8 or 12 h co-culture, which was significantly reduced when HBE was transfected with HIF-1α siRNA.
 Conclusion: Airway epithelial cells can enhance macrophages chemotaxis and pro-inflammatory cytokines expressions under hypoxic condition. HIF-1α and Cav-1 may be the important mediators in these processes.

Tumour suppressive function of HUWE1 in thyroid cancer.

HUWE1 (the HECT, UBA, and WWE domain-containing protein 1) is an ubiquitin E3 ligase which plays an important role in coordinating diverse cellular processes. It has been found to be dysregulated in various cancer type and its functions in tumorigenesis remain controversial. The potential tumour suppressive role of HUWE1 in thyroid cancer development was investigated by knocking down HUWE1 in three authentic thyroid cancer cell lines, WRO, FTC133 and BCPAP, followed by various functional assays, including cell proliferation, scratch wound healing and invasion assays. Xenograft experiment was performed to examine in vivo tumour suppressive properties of HUWE1. Small-interfering RNA mediated knockdown of HUWE1 promoted cell proliferation, cell migration and invasion in thyroid cancer cells. Overexpression of HUWE1 conferred partial sensitivity to chemo drugs interfering with DNA replication in these cells. Moreover, HUWE1 was found to be down-regulated in human thyroid cancer tissues compared with matched normal thyroid tissues. In addition, overexpression of HUWE1 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that HUWE1 can regulate p53 protein level through its stabilization. HUWE1 functions as a tumour suppressor in thyroid cancer progression, which may represent a novel therapeutic target for prevention or intervention of thyroid cancer.

Evaluation of Serum Vascular Adhesion Protein-1 as a Potential Biomarker in Thyroid Cancer.

Vascular adhesion protein-1 (VAP-1) is a glycoprotein that mediates tissue-selective lymphocyte adhesion. The prognostic value of VAP-1 has been determined in gastric cancer. The aim of this study was to evaluate the changes and the predictive value of serum VAP-1 in patients with thyroid cancer. A total of 126 patients with thyroid nodules and 53 healthy controls participated in this study. The patients were further divided into subgroup 1 (69 cases with benign thyroid nodules) and subgroup 2 (57 cases with thyroid cancer). Serum VAP-1 was measured by time-resolved immunofluorometric assay. Diagnostic value of presurgical VAP-1 for thyroid cancer was conducted by receiver operating characteristic (ROC) curves. Serum levels of VAP-1 were significantly lower in thyroid cancer group than in healthy control and benign thyroid nodule groups. VAP-1 concentrations negatively correlated with serum thyroglobulin (Tg) levels in thyroid cancer patients (r = -0.81; p < 0.001). The optimum cut-off value of VAP-1 was 456.6 ng/mL with a 77.4% specificity and 66.7% sensitivity for thyroid cancer diagnosis. Serum VAP-1 decreased in thyroid cancer patients and VAP-1 could be a potential useful adjunct biomarker in the diagnosis of thyroid cancer.

Filamin A (FLNA) modulates chemosensitivity to docetaxel in triple-negative breast cancer through the MAPK/ERK pathway.

A previous RNA interference (RNAi) screen identified filamin A (FLNA) as a potential biomarker to predict chemosensitivity in triple-negative breast cancer (TNBC). However, its ability to modulate chemosensitivity and the underlying mechanism has not been investigated. Genetic manipulation of FLNA expression has been performed in an immortalized noncancerous human mammary epithelial cell line and four TNBC cell lines to investigate its effect on chemosensitivity. Western blot analysis was performed to identify the potential signaling pathway involved. Xenograft mouse model was used to examine the in vivo role of FLNA in modulating chemosensitivity. Overexpression of FLNA conferred chemoresistance to docetaxel in noncancerous human mammary epithelial cells. Knockdown of FLNA sensitized four TNBC cell lines, MDA-MB-231, HCC38, Htb126, and HCC1937 to docetaxel which was reversed by reconstituted FLNA expression. Decreased FLNA expression correlated with decreased activation of ERK. Constitutive activation of ERK2 reversed siFLNA-induced chemosensitization. Inhibition of MEK1 recapitulates the effect of FLNA knockdown. MDA-MB-231 xenograft with FLNA knockdown showed enhanced response to docetaxel compared with control xenograft with increased apoptosis. FLNA can function as a modulator of chemosensitivity to docetaxel in TNBC cells through regulation of the MAPK/ERK pathway both in vitro and in vivo. FLNA may serve as a novel therapeutic target for improvement of chemotherapy efficacy in TNBC.