Cell Carcinoma Of Larynx Research Articles

Bio-fabrication of pigment-capped silver nanoparticles encountering antibiotic-resistant strains and their cytotoxic effect towards human epidermoid larynx carcinoma (HEp-2) cells.

Bacterial biomolecule-mediated nanoparticle (NP) synthesis constitutes a reliable, eco-friendly approach that ameliorates green-chemistry principles. In this study, stable silver nanoparticles were synthesized by exposing aqueous silver ions to an extracellular diffusible pigment produced by Pseudomonas aeruginosa (PA6) under optimized laboratory conditions. Spectroscopic and microscopic analyses showed the typical characteristics of silver with an average size of ∼28.30 nm and spherical shape. The particles were polydispersed and showed no definite agglomeration with a zeta potential of −32.3 mV, conferring stability. Antimicrobial studies were carried out using 5, 15, 25 and 50 μg mL−1 concentrations of pcAgNPs, which showed significant antibacterial activity toward clinically important pathogens at all concentrations compared to with the control sample. The bactericidal effect induced by pcAgNPs associated with cell damage was well demonstrated using electron microscopic studies. ROS production was measured using the DCFH-DA method and the oxidative stress was assessed by measuring the reduced glutathione (GSH) levels. Cytotoxicity studies on HEp-2 (Human Epidermoid Larynx Carcinoma) cells exposed to pcAgNPs showed dose-dependent cytotoxic effect with IC50 of 14.8 μg mL−1 compared to with IC50 of 7.38 μg mL−1 for the Vero cell control. Mechanistically, the pcAgNPs activated p53 that induced catalase, leading to apoptosis and DNA fragmentation via a p53 transcriptional pathway and electron transport arrest, which resulted in cell death. This synergistic efficacy of pigment-AgNPs demonstrated excellent antimicrobial and anti-proliferative activities, providing a potential lead for developing a broad-spectrum antibacterial agent and improving the therapeutic modalities targeting carcinoma cells at the gene level.

LncRNA UCA1 promotes cell proliferation, invasion and migration of laryngeal squamous cell carcinoma cells by activating Wnt/β-catenin signaling pathway.

In view of the poor prognosis of laryngeal squamous cell carcinoma (LSCC) and the functionality of long non-coding (lnc)RNA UCA1 in different types of cancer, the present study aimed to investigate the role of UCA1 in the development and progression of LSCC. A total of 90 patients with LSCC and 90 healthy subjects were enrolled in the present study. Expression levels of UCA1 in tumor tissues and adjacent healthy tissues, as well as serum of patients with LSCC and normal controls were detected by reverse transcription-quantitative polymerase chain reaction. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic value of serum UCA1 for LSCC. Survival curves were plotted using the Kaplan-Meier method and employed to evaluate the prognosic values of serum UCA1 for LSCC. Cell proliferation, migration and invasion were detected using the cell proliferation assay, and Transwell migration and invasion assays, respectively. Expression levels of Wnt/β-catenin-associated proteins were detected by western blot analysis. Results indicated that the expression levels of UCA1 were significantly higher in tumor tissues compared with adjacent healthy tissues in the majority of patients with LSCC. In addition, serum levels of UCA1 were significantly higher in patients with LSCC coapred with healthy controls. UCA1 overexpression promoted, whereas UCA1 knockdown inhibited the proliferation, migration and invasion of LSCC cells. UCA1 overexpression activated the Wnt/β-catenin signaling pathway in LSCC cells, whereas treatment with Wnt inhibitor reduced the enhancing effects of UCA1 overexpression on the proliferation, migration and invasion of LSCC cells. The present findings suggest that UCA1 can promote cell proliferation, invasion and migration of LSCC cells by activating the Wnt/β-catenin signaling pathway.

Knockdown of DGCR5 enhances the radiosensitivity of human laryngeal carcinoma cells via inducing miR-195.

Long noncoding RNAs (lncRNAs) exert critical roles in the development of various cancers, including human laryngeal cancer. Radioresistance contributes to the predominant causes of laryngeal cancer recurrence after radiotherapy. The aim of our study was to investigate the association of dysregulated lncRNA and radiation resistance in human larynx squamous carcinoma. Here, we investigated the biological roles of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) in radioresistance of human laryngeal cancer. Two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were used in the present study. We observed that DGCR5 was significantly upregulated in Hep-2R cells. Inhibition of DGCR5 by LV-shDGCR5 transfection restrained Hep-2R cell proliferation and sensitized cells to radiation. Reversely, overexpression of DGCR5 exhibited an opposite phenomenon in vitro. In addition, microRNA (miR)-195 was predicted as a direct downstream target of DGCR5. Dual-luciferase reporter and RNA immunoprecipitation assays verified the direct interaction between them. Meanwhile, miR-195 was observed to be reduced in Hep-2R cells and miR-195 mimics repressed Hep-2 cell growth. Moreover, radiosensitivity of Hep-2R cells was greatly enhanced by overexpression of miR-195, which could be reversed by upregulation of DGCR5. Finally, in vivo experiments were used to validate that knockdown of DGCR5 suppressed laryngeal carcinoma via targeting miR-195. In conclusion, we indicated that DGCR5 could contribute to the radioresistance of human laryngeal carcinoma cells via sponging miR-195.

Combination of TPF regimen and cinobufotalin inhibits proliferation and induces apoptosis in human hypopharyngeal and laryngeal squamous cell carcinoma cells.

BackgroundHypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma have a potential risk of metastasis and local recurrence. It is urgent to determine novel effective therapy.MethodsUMSCC5 and FADU cells were treated with TPF regimen (a mixture comprising paclitaxel, cisplatin, and 5-fluorouracil), cinobufotalin, and combination. Cell counting kit-8 (CCK-8), clone formation assays, and flow cytometry were performed to investigate whether combination of cinobufotalin and TPF can exhibit synergistic effects in reducing tumor growth.ResultsCCK-8 assay and IC50 analysis showed that the TPF and cinobufotalin could suppress viability of UMSCC5 and FADU cells, implying the strong antitumor effect. The synergism between TPF and cinobufotalin was further verified by the CCK-8 and clone formation assays showing the TPF- and cinobufotalin-suppressed cell proliferation synergistically. Notably, flow cytometry showed that the combination also promoted apoptosis synergistically.ConclusionTPF regimen combining cinobufotalin suppressed proliferation and promoted apoptosis synergistically in hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma cells.

Luminescent silica mesoparticles for protein transduction

Unlike silica nanoparticles, the potential of silica mesoparticles (SMPs) (i.e. particles of submicron size) for biological applications in particular the in vitro (let alone in vivo) cellular delivery of biological cargo has so far not been sufficiently studied. Here we examine the potential of luminescent (namely, octahedral molybdenum cluster doped) SMPs synthesised by a simple one-pot reaction for the labelling of cells and for protein transduction into larynx carcinoma (Hep-2) cells using GFP as a model protein. Our data demonstrates that the SMPs internalise into the cells within half an hour. This results in cells that detectably luminesce via conventional methods. In addition, the particles are non-toxic both in darkness and upon photo-irradiation. The SMPs were modified to allow their functionalisation by a protein, which then delivered the protein (GFP) efficiently into the cells. Thus, the luminescent SMPs offer a cheap and trackable alternative to existing materials for cellular internalisation of proteins, such as the HIV TAT protein and commercial protein delivery agents (e.g. Pierce™).

From Photoinduced to Dark Cytotoxicity through an Octahedral Cluster Hydrolysis

Octahedral molybdenum and tungsten clusters have potential biological applications in photodynamic therapy and bioimaging. However, poor solubility and hydrolysis stability of these compounds hinder their application. The first water-soluble photoluminescent octahedral tungsten cluster [{W6 I8 }(DMSO)6 ](NO3 )4 was synthesised and demonstrated to be at least one order of magnitude more stable towards hydrolysis than its molybdenum analogue. Biological studies of the compound on larynx carcinoma cells suggest that it has a significant photoinduced toxicity, while the dark toxicity increases with the increase of the degree of hydrolysis. The increase of the dark toxicity is associated with the in situ generation of nanoparticles that clog up the cisternae of rough endoplasmic reticulum.

Inhibition of GLUT-1 expression and the PI3K/Akt pathway to enhance the chemosensitivity of laryngeal carcinoma cells in vitro.

BackgroundThe mechanism of chemoresistance remains unknown. Here, we investigated if glucose transporter-1 (GLUT-1) and PI3K/Akt pathways are associated with the sensitivity to cisplatin in Hep-2 laryngeal carcinoma cells and whether the inhibition of GLUT-1 and the PI3K/Akt pathways enhances the chemosensitivity of Hep-2 cells.MethodThe effects of inhibiting GLUT-1 by a GLUT-1 siRNA, and PI3K/Akt by Ly294002, on cisplatin-induced effects were assessed in vitro.ResultsGLUT-1 siRNA and cisplatin showed a synergistic effect in inhibiting the proliferation of Hep-2. LY294002 and cisplatin also showed a synergistic effect in inhibiting the proliferation of Hep-2. GLUT-1 siRNA, LY294002 and cisplatin effectively inhibited the mRNA expressions and protein expressions of GLUT-1, Akt, PI3k and HIF-1α in Hep-2 cells. Furthermore, GLUT-1 siRNA and cisplatin demonstrated a synergism to inhibit the mRNA expression of HIF-1α. Moreover, it was found in this study that GLUT-1 siRNA, LY294002 and cisplatin induced the suppression of the cell cycle at G1/G2 and the increasing of apoptosis in Hep-2 cells.ConclusionThis study showed that inhibiting GLUT-1, by a GLUT-1 siRNA and inhibiting PI3K/Akt by Ly294002, could suppress the proliferation of Hep-2 alone and together with cisplatin synergistically, which demonstrated the potentials to treat laryngeal carcinoma in the future therapy. Additionally, the synergistic effect between LY294002 and cisplatin to suppress the proliferation of Hep-2 might not be from GLUT-1, Akt, PI3k and HIF-1α; the synergistic effect between GLUT-1 siRNA and cisplatin to suppress the proliferation of Hep-2 might not be from GLUT-1, Akt and PI3k and might be more or less related to HIF-1α.

Hydrogen peroxide-induced mitophagy contributes to laryngeal cancer cells survival via the upregulation of FUNDC1.

The purpose of our study was to investigate an underlying mechanism that hydrogen peroxide-induced mitophagy contributed to laryngeal cancer cells survivals under oxidative stress condition. Tumor tissue and serum samples were collected from patients with laryngeal cancer. The Hep2 cell, a human laryngeal carcinoma cell, was used in in vitro experiments. The levels of lipid peroxidation were analyzed by ELISA. Knockdown of FUNDC1 was performed by RNAi. The changes of target proteins were determined by qRT-PCR and western blot. The cells were analyzed for changes in proliferation using cell counting kit-8 and mitophagy by the mitochondrial membrane potential assay and transmission electron microscopy. FUNDC1 in laryngeal cancer tissues were relative to the levels of lipid peroxidation in laryngeal cancer patients, which suggested that FUNDC1 was associated with the status of oxidative stress in the laryngeal cancer patients. Hydrogen peroxide significantly induced the elevation of FUNDC1, a mitophagic factor, in a time- and dose-dependent manner in laryngeal cancer cells, which was dependent on ERK signal activation. Knockdown of FUNDC1 by the siRNA attenuated the survival of laryngeal cancer cells under hydrogen peroxide stimulation. Moreover, the elevated FUNDC1 was required for the occurrence of mitophagy under hydrogen peroxide stimulation, which was identified by transmission electron microscopy, the alterations of mitochondrial permeability transition and the specific mitochondrial protein, hsp60. Inhibition of mitophagy with cyclosporine A could also effectively attenuate the laryngeal cancer cells survival under hydrogen peroxide stimulation. Hydrogen peroxide upregulated the expression of FUNDC1 through the activation of ERK1/2 signal to trigger a mitophagic response, giving laryngeal cancer cells a befit for survival. These findings suggested that FUNDC1 might be a potential target for the treatment of laryngeal cancer accompanied with high lipid peroxidation status.

Investigation of the diagnostic value of anti-dense fine speckled 70/lens epithelium derived growth factor p75 autoantibody for autoimmune diseases

Antinuclear antibodies (ANA) are the autoantibodies that are produced against nuclear antigens in the cell nucleus and/or cytoplasm, and are one of the important diagnostic criteria in systemic autoimmune rheumatic diseases (SARD). Until today, several methods have been developed for detecting ANA's. However, indirect immunofluorescence (IIF) technique, that is also known as one of the oldest methods, is still the most commonly used one. Typically, anti-dense fine speckled 70/Lens epithelium derived growth factor p75 (anti-DFS70/LEDGF p75) autoantibody can be detected via IIF method where in HEp-2 (human larynx carcinoma) cells are used. The dense fine speckled (DFS) pattern method can be masked and remain unnoticed by the IIF method when it exists with the other ANA. Anti-DFS70 autoantibodies seldomly appear in SARD patients compared to healthy individuals. Moreover, these antibodies may appear in different chronic inflammatory conditions like interstitial cystitis, chronic fatigue syndrome, atopic dermatitis and Vogt-Koyanagi-Harada syndrome. The aim of this study was to determine the frequency of anti-DFS70 autoantibodies by immunblot (IB) method in patients sera with and without DFS70 staining pattern by IIF and to determine if the presence of anti-DFS70 has a clinical impact when included in ANA testing algorithm. In our study, a total of 60 patients' sera in which DFS pattern was defined by IIF method and 67 patients' sera in which other patterns observed were included in the study and anti-DFS70 autoantibody was investigated by IB method in these sera. In 67 patient samples which have shown the other patterns three (4.5%) samples were determined to be anti-DFS70 positive by IB. In 55 patients who were determined to have IIF-DFS pattern (+)/IB anti-DFS70 (+), 6 (11%) were diagnosed as SARD and the other antinuclear antibodies (ANA) were found as negative by IB. In the other group with the other ANA patterns detected, none of the SARD-diagnosed 22 patients had shown anti-DFS70 by IB method. Sixteen (26.6%) samples in the group that was positive for the IIF-DFS pattern were obtained from rheumatology and physical therapy and rehabilitation clinics, 32 (47.7%) samples were from the group in which other patterns observed and were also obtained from those clinics. DFS pattern was detected significantly more frequent in the samples from other clinics in comparison to the samples from rheumatology and physical therapy and rehabilitation clinics (p= 0.018). In our study, it was concluded that DFS pattern can be defined by IIF method by only specialists, however, since homogeneous-like and mixed patterns can be confused especially in low titers, there is a need for a second well-validated immunological test that could detect anti-DFS70 auto-antibody.

P2-004 - Autophagy inhibition improves CD47-blocking immunotherapy in laryngeal squamous cell carcinoma

Background: CD47 blocking immunotherapy, an effective treatment strategy that triggers phagocytosis of tumor cells by macrophage in diverse cancers. Here, we investigated the synergistic antitumor effect of simultaneously targeting CD47 and autophagy in Laryngeal squamous cell carcinoma (LSCC) cells. Methods: Cell vitality was detected by MTT assay. Autophagic flux was analyzed by laser scanning confocal microscope. Protein expression was examined by western blotting. In vivo anti-tumor activity was assessed using LSCC xenograft established in nude mice. Results: We demonstrated that blocking CD47-SIRPα by SIRPα-Fc could significantly induce cytotoxicity and phagocytosis of LSCC cells. Meanwhile, we found that autophagy was activated by SIRPα-Fc, inducing the activity of LC3 which was converted from LC3-I to LC3-II, while Akt/mTOR signaling pathway was involved in autophagic process. Furthermore, inhibiting SIRPα-Fc-induced autophagy by pharmacological inhibitors or small interfering RNA significantly enhanced SIRPα-Fc-triggered macrophage phagocytosis and cytotoxicity, revealing autophagy played a cytoprotective role in CD47-targeted therapy of LSCC. Moreover, xenograft experiment showed that combination of autophagy inhibitor potentiated the anti-tumor efficacy of SIRPα-Fc, with recruitment of macrophages and apoptosis of tumor cells. Conclusion: These findings demonstrated that CD47-blocking therapies alone and in combination with autophagy inhibitor could potently kill LSCC cells, highlighting potential therapeutic strategies for cancer immunotherapy.

Genetic characteristics of CV-A12 VP1 region and clinical manifestations of CV-A12-associated severe hand, foot and mouth disease in Qingdao

Objective To investigate the molecular characteristics of coxsackievirus A12 (CV-A12) and to understand the clinical manifestations of severe hand, foot and mouth disease (HFMD) caused by CV-A12 in Qingdao. Methods Throat swabs of HFMD, herpangina and influenza-like cases from 2011 to 2016 were detected for enteroviruses (EVs) in Qingdao. Human rhabdomyosarcoma (RD) and human laryngeal carcinoma (Hep-2) cells were used for virus proliferation and CV-A12 strains were identified through a semi-nest RT-PCR. The full-length of VP1 gene of CV-A12 strains was sequenced and phylogenetically analyzed using MEGA7.0 software package. Clinical data of severe HFMD cases positive for CV-A12 were collected and analyzed. Results CV-A12-positive HFMD, herpangina and influenza-like cases accounted for 0.3%(18/6 798), 1.2%(2/169) and 0.1%(1/676) in Qingdao, respectively. Most of the HFMD caused by CV-A12 in children were mild before 2013 (84.6%, 11/13), while hospitalized severe cases with neurological symptoms (100%, 5/5) became more common after 2013. Phylogenetic analysis of the VP1 region revealed that CV-A12 strains worldwide could be divided into two genotypes, A and B. All of the CV-A12 strains successfully sequenced in Qingdao from 2011 to 2016 belonged to genotype B, and 88.9% (16/18) of them belonged to subgenotype B2. All hospitalized severe cases of CV-A12-caused HFMD after 2013 were associated with strains in branch B2b of subgenotype B2. Conclusion CV-A12 was one of the pathogens causing HFMD, herpangina and influenza-like illness in children in Qingdao. Strains of genotype B2 were the predominant CV-A12 strains circulating in Qingdao in recent years. CV-A12-caused HFMD might complicated by nervous system damage. Key words: Coxsackievirus A12; Severe hand, foot and mouth disease; Phylogenetic analysis

MiR-140-5p inhibits larynx carcinoma invasion and angiogenesis by targeting VEGF-A.

Larynx carcinoma is a common head-neck malignant tumor. Recent investigations showed the involvement of microRNA (miR) in regulation of multiple tumors. miR-140-5p showed decreased expression in various cancers, but without knowledge regarding its expression in larynx carcinoma and effects on cell invasion and angiogenesis. Real-time quantitative PCR was firstly employed to measure miR-140-5p expression in larynx carcinoma and controlled tumor adjacent tissues. In larynx cancer cell line, agomir or antagomir of miR-140-5p was applied to up-regulate or down-regulate miR-140-5p, respectively. Western blot was used to evaluate vascular endothelial growth factor A (VEGF-A) expression, and cell proliferation was modified by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT) approach. Transwell approach was used to measure cell invasion, and angiogenesis assay was used to detect the effect on angiogenesis. Luciferase report assay (LRA) measured targeting binding between miR-140-5p and VEGF-A. Comparing to tumor adjacent tissues, larynx carcinoma cells showed significantly decreased miR-140-5p expression. Agomir up-regulated miR-140-5p expression and weakened proliferation and invasion potency, and inhibited angiogenesis. Antagomir down-regulated miR-140-5p and presented the opposite results. Finally, LRA confirmed direct binding between miR-140-5p and VEGF-A. MiR-140-5p can target VEGF-A in larynx carcinoma cell line to inhibit cell invasion and angiogenesis. MiR-140-5p thus may work as the direct molecular target of larynx carcinoma.

MicroRNA-138 inhibits proliferation and induces apoptosis of laryngeal carcinoma via targeting MAPK6.

To explore whether microRNA-138 could regulate the incidence and progression of laryngeal carcinoma through modulating proliferation and apoptosis of laryngeal carcinoma cells via MAPK6. MicroRNA-138 expression in laryngeal carcinoma tissues and paracancerous tissues were detected by qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The regulatory effects of microRNA-138 on proliferation and apoptosis of laryngeal carcinoma cells were detected by colony formation assay and flow cytometry, respectively. Target gene of microRNA-138 was predicted by online software and verified by luciferase reporter gene assay. Corresponding plasmids of microRNA-138 and the target gene were constructed. Rescue experiments were conducted to explore the regulatory effect of microRNA-138 on the target gene. MicroRNA-138 was downregulated in laryngeal carcinoma tissues than that of paracancerous tissues. MicroRNA-138 knockdown resulted in increased proliferation and decreased apoptosis of laryngeal carcinoma cells. MAPK6 was predicted as the target gene of microRNA-138. Luciferase reporter gene assay further verified that MAPK6 could directly bind to microRNA-138. Both mRNA and protein levels of MAPK6 were downregulated after microRNA-13 overexpression in laryngeal carcinoma cells. Rescue experiment results indicated that increased proliferation and decreased apoptosis of laryngeal carcinoma cells resulted from microRNA-13 knockdown were partially reversed by MAPK6 overexpression. MicroRNA-138 is downregulated in laryngeal carcinoma patients. MicroRNA-138 knockdown promotes proliferation and inhibits apoptosis of laryngeal carcinoma cells via inhibiting MAPK6 expression.

Characterization of selective exosomal microRNA expression profile derived from laryngeal squamous cell carcinoma detected by next generation sequencing.

Exosomes are nanometer-scale extracellular vesicles derived from almost all types of cells and key signaling mediators between cancer cells and their microenvironment. Certain microRNAs (miRNAs) are selected for exosome packing and exclusion from parental cells, while other miRNAs are selectively retained by cells, suggesting a biological role for these miRNAs in tumor malignant progression. In the present study, we isolated and characterized the exosomes derived from the laryngeal squamous cell carcinoma (LSCC) cell line AMC-HN-8 for the first time, and identified a subset of miRNAs enriched in the exosomes compared with parental cells, such as miR-1246, miR-1290, miR-335-5p, miR-127-3p and miR-122-5p through small RNA sequencing combined with reverse transcription-quantitative PCR (RT-qPCR) analysis. Potential target prediction, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed possible functions associated with these selective exosomal miRNAs. In conclusion, the present study demonstrated that the LSCC cell line AMC-HN-8 can release exosomes and cells can selectively pack certain miRNAs into exosomes.

MicroRNA-1225-5p acts as a tumor-suppressor in laryngeal cancer via targeting CDC14B.

This study aimed to investigate the role of miRNA-1225-5p (miR-1225) in laryngeal carcinoma (LC). We found that the expression of miR-1225 was suppressed in human LC samples, while CDC14B (cell division cycle 14B) expression was reinforced in comparison with surrounding normal tissues. We also demonstrated that enhanced expression of miR-1225 impaired the proliferation and survival of LC cells, and resulted in G1/S cell cycle arrest. In contrast, reduced expression of miR-1225 promoted cell survival. Moreover, miR-1225 resulted in G1/S cell cycle arrest and enhanced cell death. Further, miR-1225 targets CDC14B 3'-UTR and recovery of CDC14B expression counteracted the suppressive influence of miR-1225 on LC cells. Thus, these findings offer insight into the biological and molecular mechanisms behind the development of LC.

Long non-coding RNA AFAP1-AS1/miR-320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance.

AFAP1‐AS1 is a long non‐coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1‐AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1‐AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp‐2 cells. Indeed, we found not only that AFAP1‐AS1 was up‐regulated in laryngeal carcinoma specimens and cells, but also that stemness‐associated genes were overexpressed. Silencing of AFAP1‐AS1 promoted HEp‐2 cell chemoresistance under cisplatin treatment. Expression of AFAP1‐AS1 was increased in drug‐resistant Hep‐2 cells. We then probed the mechanism of AFAP1‐AS1 activity and determined that miR‐320a was a potential molecular target of AFAP1‐AS1. Luciferase reporter and qRT‐PCR assays of AFAP1‐AS1 and miR‐320a levels in human specimens and cell cultures indicated that AFAP1‐AS1 negatively regulates miR‐320a. To discover the molecular mechanism of miR‐320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3′‐untranslated regions (3′‐UTR) of RBPJ into a luciferase reporter, we determined that miR‐320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1‐AS1 increases RBPJ expression by negatively regulating miR‐320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1‐AS1 silencing. Taken together, these results suggest that AFAP1‐AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR‐320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.

Overexpression of Beclin1 inhibits proliferation and promotes apoptosis of human laryngeal squamous carcinoma cell Hep-2.

ObjectiveBeclin1 was previously found to be downregulated in human laryngeal cancer (LC) tissues, and it results in poor prognosis. This study aimed to further confirm the antitumor effects of Beclin1 in LC cell line Hep-2.Materials and methodsBeclin 1 was overexpressed in Hep-2 cells using liposomal transfection and confirmed using reverse transcription polymerase chain reaction and Western blotting. Then, cell proliferation and apoptosis were determined in control (untransfected), empty vector transfected, and Beclin1 overexpressed groups using MTT and flow cytometry procedure, respectively.ResultsThe expression of the Beclin1 gene in Hep-2 cells was significantly increased after vector transfection compared with control (1.173±0.046 vs 0.453±0.016, P<0.01) and empty vector (1.173±0.046 vs 0.440±0.021, P<0.01). Overexpression of Beclin1 inhibited proliferation at 4 days (0.619±0.051 vs 0.891±0.081 and 0.619±0.051 vs 0.878±0.105, P<0.01), 5 days (0.684±0.078 vs 1.127±0.094 and 0.684±0.078 vs 1.162±0.117, P<0.01), and 6 days (0.725±0.069 vs 1.168±0.103 and 0.725±0.069 vs 1.194±0.097, P<0.01) and promoted apoptosis (14.48%±1.42% vs 4.07%±0.66% and 14.48%±1.42% vs 4.39%±0.80%, P<0.01) in Hep-2 cells in comparison with the control and empty vector groups, respectively.ConclusionBeclin1 may be an underlying target for the treatment of LC. This study has provided some experimental basis for the gene therapy of LC.

Synthesis and structures of CuI,II complexes with a 2,2´-bipyridine derivative bearing a (+)-3-carene moiety

The complex salt {[CuL2][Cu4I6]•MeCN}n (1) and the compound [Cu4L3I4]•3 MeCN (2) (L is a chiral ligand bearing a natural monoterpene (+)-3-carene moiety) were synthesized. The crystal structures of compounds 1 and 2 were determined by X-ray diffraction. The structure of compound 1 consists of complex cations [CuL2]2+ (N3O2 polyhedron is a trigonal bipyramid) and CuI coordination polymers (CuI4 polyhedron is a tetrahedron) as anions. The experimental magnetic moment μeff at 300 K is 1.90 μB, which is consistent with the X-ray diffraction data and the assumption that compound 1 is mixed-valence. The structure of compound 2 comprises a tetranuclear CuI complex, in which three Cu atoms are coordinated by two N atoms of the ligand L and two I atoms, and the fourth Cu atom is coordinated by four I atoms (coordination polyhedra are distorted tetrahedra). Compounds L and 2 were found to influence the viability of human laryngeal carcinoma cells (Hep2). The IC50 value for complex 2 (13.0±1.7 μM) is substantially smaller than IC50 for compound L (30.5±0.5 μM).

PO-093 A hep-2 cell model as tool for analysing DNA double strand breaks in cancer patients

IntroductionDNA double strand breaks (DSB) are one of the most severe DNA damages and as thus can lead to genomic instability favouring cancer. The number of phosphorylated histone variant H2AX (γH2AX) correlates with the number of DSB and can be quantitatively detected by immunofluorescence imaging. In particular, we are interested on the individual response and adverse effects of DSB–inducing chemotherapeutic drugs such as etoposide in the context of the chemoresistance–inducing and tumour–associated antigen dense fine speckled protein of 70 kDa (DFS–70).Material and methodsHEp-2 wild type and CRISRP/Cas9-generated DFS-70 knock-out (DFS-70 KO) cells were treated with the topoisomerase inhibitor etoposide (0–10 µM) to induce DSB. The AKLIDES NUK system (Medipan GmbH, Dahlewitz, Germany), a fully automated and standardised immunofluorescence imaging and data processing platform, was used to measure the number of γH2AX foci and nucleus size. Additionally, immunoblotting was performed to analyse yH2AX.Results and discussionsBoth the protein analysis by Western blotting and Immunofluorescence analysis by AKLIDES NUK system showed an elevated formation of γH2AX with increased etoposide concentration. HEp-2 wild type cells indicated a significant induction (p<0.01) of DSB up to 13 foci per cell after 16 hours of etoposide treatment. Adverse effects could be observed on nucleus size that increased significantly upon treatment in all cell lines. No difference in the basal level of γH2AX foci were detectable comparing wild type and DSF-70 KO cells. In contrast, two out of five DFS-70 KO clones exhibited an increased foci number in comparison to the wild type control after 16 hours of etoposide treatment. This finding might indicate that DSF-70 KO sublines are more sensitive to topoisomerase inhibition than HEp–2 wild type cells.ConclusionThe automated multiparameter imaging platform AKLIDES allows an individual and differential detection of γH2AX foci formation and nuclei size in human laryngeal carcinoma cells, HEp–2 and sublines. Therefore, this system can be useful to identify cancer progression e.g. analysing peripheral blood mononuclear cells of patients with malignant lymphoma. Moreover, additional biomarkers defining cancer progression can be integrated into the analysis.

Polycaprolactone nanofibres loaded with 20(S)-protopanaxadiol for in vitro and in vivo anti-tumour activity study.

In this work, 20(S)-protopanaxadiol (PPD)-loaded polycaprolactone (PCL) nanofibres were successfully fabricated by the electrospinning technique using Tween 80 as a solubilizer. Firstly, smooth and continuous nanofibres were collected using suitable solvents and appropriate spinning conditions. Secondly, nanofibre mats were characterized by scanning electron microscopy, thermogravimetric (TG) analysis, Fourier transform infrared spectroscopy and mechanical testing. Finally, nanofibrous membranes were evaluated using water contact angle, in vitro drug release, biodegradation test, in vitro and in vivo anti-tumour activity and cell apoptosis assay. Scanning electron microscopic observations indicated that the diameter of the drug-loaded nanofibres increased with the increase of drug concentration. TG analysis and mechanical test showed that nanofibres were equipped with great thermal and mechanical properties. Biodegradation test exhibited that the structure of fabricated nanofibres had a certain degree of change after 15 days. An in vitro release study showed that PPD from drug-loaded nanofibres could be released in a sustained and prolonged mode. The cytotoxic effect of drug-loaded nanofibre mats examined on human laryngeal carcinoma cells (Hep-2 cells) demonstrated that the prepared nanofibres had a remarkable anti-tumour effect. Meanwhile, the drug-loaded fibre mats showed a super anti-tumour effect in an in vivo anti-tumour study. All in all, PCL nanofibres could be a potential carrier of PPD for cancer treatment.