Human Laryngeal Carcinoma Cells Research Articles

Nanosized mesoporous metal–organic framework MIL-101 as a nanocarrier for photoactive hexamolybdenum cluster compounds

Inclusion compounds of photoluminescent hexamolybdenum cluster complexes in the chromium terephthalate metal-organic framework, MIL-101 (MIL, Matérial Institut Lavoisier) were successfully synthesized in two different ways and characterized by means of powder X-Ray diffraction, chemical analysis and nitrogen sorption. Some important functional properties of hexamolybdenum cluster complexes for biological and medical applications, in particular singlet oxygen generation ability, luminescence properties, cellular uptake behavior and cytotoxicity were studied. It was revealed that the inclusion compounds possessed significant singlet oxygen generation activity. The materials obtained showed a low cytotoxicity, thus allowing them to be used in living cells. Confocal microscopy of human larynx carcinoma (Hep-2) cells incubated with the inclusion compounds showed that MIL-101 performed as a nanocarrier adhering to the external cell membrane surface and releasing the cluster complexes which that penetrated into the cells. Moreover, photoinduced generation of reactive oxygen species (ROS) in Hep-2 cells incubated with inclusion compounds was demonstrated. The cluster supported on MIL-101 was shown to possess in vivo phototoxicity.

MicroRNA-133a suppresses the proliferation, migration, and invasion of laryngeal carcinoma cells by targeting CD47.

The study aims to investigate the possible mechanisms of microRNA-133a (miR-133a) targeting CD47 on cell proliferation, apoptosis, migration, and invasion in laryngeal carcinoma. Forty-two laryngeal carcinoma tissue specimens confirmed by pathological examination from laryngeal carcinoma patients as the case group were collected, and 20 chronic laryngitis tissues were gathered as the control group. The human laryngeal carcinoma cell line Hep-2 was marked as the miR-133a mimic, negative control (NC), miR-133a inhibitor, CD47-siRNA, miR-133a inhibitor + CD47-siRNA, and Mock groups. The expression of CD47 protein and miR-133a was detected by immunohistochemistry (IHC) and qRT-PCR. Dual luciferase assay system was used to determine the relationship between CD47 and miR-133a. Western blotting was used to measure the protein expression of CD47 and miR-133a. 5-Ethynyl-2'-deoxyuridine (EDU) method was used to detect the cell proliferation, and flow cytometry and Transwell were used to measure the cell apoptosis and migration and invasion, respectively. The miR-133a expression in laryngeal carcinoma tissues was significantly lower, while the CD47 expression was higher than that in chronic laryngitis tissues (both P<0.01). The expression of miR-133a in the miR-133a mimic group was significantly higher than that in other groups (P<0.05), and the messenger RNA (mRNA) and protein expression of CD47 in the CD47-siRNA and miR-133a mimic groups were significantly lower than those in the Mock and NC group (all P<0.05), while the mRNA and protein expression of CD47 in the miR-133a inhibitor group were higher than in other groups (all P<0.05). After transfection, the CD47-siRNA group had the strongest inhibitory activity, while the number of living cells in the miR-133a inhibitor group was significantly higher than that in other groups (all P<0.05). The apoptosis rates in the miR-133a mimic and CD47-siRNA groups were significantly higher than that in the Mock and NC groups (all P<0.05). The cell numbers that penetrated membrane in the miR-133a mimic and CD47-siRNA groups were less than in the Mock and NC groups (all P<0.05). Upregulated miR-133a could inhibit proliferation, invasion, and migration and promote cell apoptosis in laryngeal carcinoma by targeting CD47. miR-133a targeting CD47 could be a new direction in the diagnosis and treatment of laryngeal carcinoma.

Effect of silencing key proteins in telomerase mechanism and alternative lengthening of telomeres mechanism in laryngeal cancer cells

PurposeTo explore the influences of telomerase and alternative lengthening of telomeres mechanism on telomere length and laryngeal squamous cell carcinoma in vitro and in vivo. Materials and methodsShort hairpin RNA expression vectors targeting the messenger TERT, TRF2, RAD51 and NBS1 were constructed. The mRNA and protein expression of targeted genes in human laryngeal squamous carcinoma cell line HEp-2 was evaluated by reverse transcription polymerase chain reaction and Western blotting separately. The length of telomere was analyzed by fluorescent in-situ hybridization. Cell viability was examined by cell counting Kit-8. Effects on tumor growth were also investigated in vivo. ResultsThe transfection of multiple short hairpin RNAs expression plasmid significantly inhibited the mRNA and protein expression of related genes. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes resulted in the shortening of telomere length in HEp-2 cell. However, silence of alternative lengthening of telomeres mechanism related genes could shorten the telomere length but had no significant difference. Both simultaneously and separately blocking telomerase mechanism and alternative lengthening of telomeres mechanism resulted in reduction of tumor cell viability. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes inhibited the tumor growth in vivo. ConclusionsThe inhibition of telomere related gene may be a promising strategy for the treatment of laryngeal squamous cell carcinoma.

Zinc phthalocyanine-conjugated with bovine serum albumin mediated photodynamic therapy of human larynx carcinoma

Phthalocyanines, which are classified as second-generation photosensitizers, have advantageous photophysical properties, and extensive studies have demonstrated their potential applications in photodynamic therapy. The present work describes the preparation of a new zinc phthalocyanine conjugated to bovine serum albumin (compound 4a) and its photodynamic efficiency in human larynx-carcinoma cells (HEp-2 cells). The unconjugated precursor (compound 4) was also studied. Compounds 4 and 4a penetrated efficiently into the cell, exhibiting cytoplasmic localization, and showed no cytotoxicity in the dark. However, high photodynamic activities were observed in HEp-2 cells after treatments with 5 µM photosensitizers and 4.5 J cm−2 light. These conditions were sufficient to decrease the cell viability to 57.93% and 32.75% for compounds 4 and 4a, respectively. The present results demonstrated high photodynamic efficiency of zinc phthalocyanine conjugated with bovine serum albumin in destroying the larynx-carcinoma cells.

Effect of multidrug resistance 1/P-glycoprotein on the hypoxia-induced multidrug resistance of human laryngeal cancer cells.

In a previous study, it was demonstrated that hypoxia upregulated the multidrug resistance (MDR) of laryngeal cancer cells to chemotherapeutic drugs, with multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp) expression also being upregulated. The present study aimed to investigate the role and mechanism of MDR1/P-gp on hypoxia-induced MDR in human laryngeal carcinoma cells. The sensitivity of laryngeal cancer cells to multiple drugs and cisplatin-induced apoptosis was determined by CCK-8 assay and Annexin-V/propidium iodide staining analysis, respectively. The accumulation of rhodamine 123 (Rh123) in the cells served as an estimate of drug accumulation and was evaluated by flow cytometry (FCM). MDR1/P-gp expression was inhibited using interference RNA, and the expression of the MDR1 gene was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. As a result, the sensitivity to multiple chemotherapeutic agents and the apoptosis rate of the hypoxic laryngeal carcinoma cells increased following a decrease in MDR1/P-gp expression (P<0.05). Additionally, FCM analysis of fluorescence intensity indicated that the downregulated expression of MDR1/P-gp markedly increased intracellular Rh123 accumulation (P<0.05). Such results suggest that MDR1/P-gp serves an important role in regulating hypoxia-induced MDR in human laryngeal carcinoma cells through a decrease in intracellular drug accumulation.

Cholesterol-modified poly(lactide-co-glycolide) nanoparticles for tumor-targeted drug delivery

Poly(lactide-co-glycolide)-cholesterol (PLGA-C)-based nanoparticles (NPs) were developed for the tumor-targeted delivery of curcumin (CUR). PLGA-C/CUR NPs with ∼200nm mean diameter, narrow size distribution, and neutral zeta potential were fabricated by a modified emulsification-solvent evaporation method. The existence of cholesterol moiety in PLGA-C copolymer was confirmed by proton nuclear magnetic resonance (1H NMR) analysis. In vitro stability of developed NPs after 24h incubation was confirmed in phosphate buffered saline (PBS) and serum media. Sustained (∼6days) and pH-responsive drug release profiles from PLGA-C NPs were presented. Blank PLGA and PLGA-C NPs exhibited a negligible cytotoxicity in Hep-2 (human laryngeal carcinoma) cells in the tested concentration range. According to the results of flow cytometry and confocal laser scanning microscopy (CLSM) studies, PLGA-C NPs presented an improved cellular accumulation efficiency, compared to PLGA NPs, in Hep-2 cells. Enhanced in vivo tumor targetability of PLGA-C NPs, compared to PLGA NPs, in Hep-2 tumor-xenografted mouse model was also verified by a real-time near-infrared fluorescence (NIRF) imaging study. Developed PLGA-C NPs may be a candidate of efficient and biocompatible nanosystems for tumor-targeted drug delivery and cancer imaging.

Combinatorial effects of geopropolis produced by Melipona fasciculata Smith with anticancer drugs against human laryngeal epidermoid carcinoma (HEp-2) cells.

The identification of natural products exerting a combined effect with therapeutic agents could be an alternative for cancer treatment, reducing the concentration of the drugs and side effects. Geopropolis (Geo) is produced by some stingless bees from a mixture of vegetable resins, gland secretions of the bees and soil. It has been used popularly as an antiseptic agent and to treat respiratory diseases and dermatosis. To determine whether Geo enhances the anticancer effect of carboplatin, methotrexate and doxorubicin (DOX), human laryngeal epidermoid carcinoma (HEp-2) cells were treated with Geo alone or in combination with each drug. Cell growth, cytotoxicity and apoptosis were evaluated using 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release, and flow cytometry. Scratch assay was used to analyze cell migration and transmission electron microscopy to observe morphologic alterations. The influence of Geo on drug resistance was also investigated assessing P-glycoprotein (P-gp) action. Geo inhibited cell proliferation and migration. The combination Geo+DOX led to the highest cytotoxic activity and induced apoptosis, leading to loss of membrane integrity. Geo had no effect on P-gp-mediated efflux of DOX. Data indicate that Geo combined with DOX could be a potential clinical chemotherapeutic approach for laryngeal cancer treatment.

Enhanced CT imaging of human laryngeal squamous carcinoma and indirect CT lymphography imaging using PEGylated PAMAM G5·NH2-entrapped gold nanoparticles as contrast agent

Abstract We report the utilization of dendrimer-entrapped gold nanoparticles (Au DENPs) modified by polyethylene glycol (PEG) with good biocompatibility for enhanced computed tomography (CT) imaging of human laryngeal squamous carcinoma and indirect CT lymphography imaging in New Zealand rabbits. In this work, PEG-modified amine-terminated poly(amidoamine) dendrimers of generation 5 (G5·NH2) were used as templates to synthesize Au DENPs, followed by acetylation of the remaining dendrimer terminal amines to generate PEGylated Au DENPs. The formed PEGylated Au DENPs was used for both enhanced CT imaging of human laryngeal squamous carcinoma cells (Hep-2 cells) and the xenograft tumor mode, and indirect CT lymphography imaging in New Zealand rabbits. In vitro cytotoxicity assay, flow cytometry analysis, and cell morphology observation revealed that the formed PEGylated Au DENPs were non-cytotoxic at a Au concentration up to 400 μM for 24 h and indicated their good biocompatibility. Transmission electron microscopy data confirmed that the PEGylated Au DENPs was uptaken dominantly by the lysosomes of the cells. The PEGylated Au DENPs enabled not only enhance CT imaging of a xenograft tumor model in nude mice after intravenous injection of the particles, but also effective indirect CT lymphography imaging in rabbits. These findings suggest that the designed PEGylated Au DENPs can be used as a potentially effective contrast agent for CT imaging of various biological systems and different kinds of carcinoma, especially lymphatic mapping and human laryngeal squamous carcinoma.

Stabilization of G-quadruplex DNA and inhibition of Bcl-2 expression by a pyridostatin analog

The G-quadruplexes located in the P1 promoter of B-cell lymphoma-2 (Bcl-2) gene are implicated to regulate Bcl-2 expression. Here, we designed a new pyridostatin analog named PDF, which exhibited high specificity and stabilizing effect toward G-quadruplexes. The luciferase assay demonstrated that PDF could significantly suppress Bcl-2 transcriptional activation in human laryngeal squamous carcinoma cells (Hep-2) cells. Besides, PDF also induced cell apoptosis in vitro assays. These results provide an excellent G-quadruplex specific ligand as an efficient Bcl-2 inhibitor. These results also implicate that PDF may be a potential anticancer drug to head neck cancer.

Apoptosis mechanism of taxol combined with resveratrol on human laryngeal carcinoma Hep-2 cells

Laryngeal cancer is one of the most common malignant tumors in the respiratory tumors, and its incidence ranks second highest in the respiratory tumors. Resveratrol (Res) is a kind of polyphenols, which can inhibit nucleotides can inhibit the growth of liver cancer cells, gastric cancer cells, pancreatic cells and other tumor cells by inhibiting ribonucleotide reductase in the cells. Taxol (Tax) is a kind of secondary metabolites of Taxus chinensis, which has anti-tumor activity for breast cancer, cervical cancer, ovarian cancer and other tumors by inhibiting cellular microtubule depolymerization. But at present the effects of resveratrol combined with taxol on human laryngeal carcinoma cell strain Hep-2 and their underlying molecular mechanisms are rarely reported. After human laryngeal cancer cell Hep-2 cells were processed with resveratrol (Res) and taxol (Tax), CCK-8 assay was used to evaluate the effect of these two herbs on the proliferation of cancer cells; AO/PI staining and JC-1 were used to detect Hep-1 cells apoptosis; the expression of Bax, Bcl-2, PARP, TRIB3, and XIAP genes was detected by real time quantitative PCR; the activity of caspase-3 and caspase-8 was determined with quantitative fluorescence method. The experimental results showed that compared with Tax, Res medication alone, joint group significantly enhanced inhibition of Hep-2 cells activity, decreased the dosage of Tax, increased the expression of Bax and PARP, TRIB3, reduced the expression of the Bcl-2 and XIAP, and promoted the activity of caspase-3 and caspase-8. The test results showed that compared with the single medication, combined group could significantly increase the inhibitory effect on Hep-2 cells, significantly reduce Tax dosage, increase expressions of Bax, PARP, TRIB3, reduce expressions of Bcl-2, XIAP, and promote activity of caspase-3, caspase-8. This indicated apoptosis of human laryngeal carcinoma cell strain Hep-2 may be induced with Res, Tax, and the combination of these two herbs by mitochondria pathway. It provides valuable clue for further research on combination of Res and Tax for the treatment of laryngeal cancer, and expanding the combined application of Res and Tax.

Synthesis and antitumor activity of some new pyrazolo[3,4-d]pyrimidine and pyrazolo[3,4-b]pyridine derivatives

A new series of pyrazolo[3,4-d]pyrimidine-3-carbonitrile and pyrazolo[3,4-b]pyridine-3-carbonitrile derivatives was synthesized by the reaction of 5-amino-1-tosyl-1H-pyrazole-3,4-dicarbonitrile as a key starting material with various electrophilic and nucleophilic reagents. All the newly synthesized compounds were structurally confirmed by various modern analytical methods (IR, 1H NMR and MS). All the title compounds have been evaluated for their potential cytotoxicity against human laryngeal epidermoid carcinoma cells (Hep2). Among the screened compounds, 3 and 4 exhibited the highest significant effect.

Characterization and cytotoxicity studies of thiol-modified polystyrene microbeads doped with [{Mo6X8}(NO3)6]2-(X = Cl, Br, I)

Halide octahedral molybdenum clusters [(Mo6X8)L6]n- possess luminescence properties that are highly promising for biological applications. These properties are rather dependent on the nature of both the inner ligands X (i.e. Cl, Br, or I) and the apical organic or inorganic ligands L. Herein, the luminescence properties and the toxicity of thiol-modified polystyrene microbeads (PS-SH) doped with [(Mo6X8)(NO3)6]2- (X=Cl, Br, I) were studied and evaluated using human epidermoid larynx carcinoma (Hep2) cell cultures. According to our data, the photoluminescence quantum yield of (Mo6I8)@PS-SH is significantly higher (0.04) than that of (Mo6Cl8)@PS-SH (6Br8)@PS-SH (6X8)@PS-SH showed that all three types of doped microbeads had no significant effect on the viability and proliferation of the cells.

Antitumor Activity of Extracts from Medicinal Basidiomycetes Mushrooms.

Aqueous extracts from the vegetative submerged mycelia of cultivated Basidiomycetes Ganoderma lucidum, Lentinus edodes, and Grifola frondosa, as well as from the fruiting bodies of G. lucidum, were found to have antitumor activity. The antitumor effect of the mycelial extracts from all 3 fungal species was ascertained in vivo in rats with implanted kidney cancer. Dystrophic changes in tumor cells and tumor necrosis (up to 90%) were noted. In vitro cytotoxicity studies of the A549 human lung adenocarcinoma cell line and HEp-2 human laryngeal epidermoid carcinoma cells showed that the extracts from the G. lucidum fruiting bodies and from the L. edodes vegetative mycelium were the most effective. The animals' immune systems were activated, and the fungal extracts displayed no toxicity when administered orally.

Studies on Biosurfactant producing bacteria from Jeppiaar salt pan with special reference to anticancer activity

Biosurfactants are surface-active compounds produced by microorganisms. These molecules reduce surface tension between aqueous solutions and hydrocarbon mixtures. In this study, we collected water samples from Jeppiaar salt pan in Chennai, Tamilnadu, India. Ten different microorganisms were isolated from salt pan water samples. Screening of biosurfactants producing potential strain was done using Hemolytic activity, Oil displacement test, Drop Collapse assay. Four isolates were positive for biosuractant production among that one isolate was found as potential biosurfactant producer. Both biochemical and 16S rRNA sequencing methods one of the potential strain was identified as Bacillus cereus JS06. The biosurfactant was extracted from cell-free broth at 72-h grown cells by step-by-step purification of acid precipitation method. The dried surfactant was used for further characterization by Fourier transmission Infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and anticancer studies. On FTIR-ATR analysis characteristic absorbance bands were observed for the extracted biosurfactant. The crude biosurfactant was screened for its anticancer activity against human laryngeal carcinoma (HEP-2) cell line and transformed aneuploid immortal keratinocyte (HeCAT) cell line. The results obtained showed that biosurfactants have better interfacial-activity and anticancer property, thus being more attractive to be applied in pharmaceuticals and medical field.

Cellular internalisation, bioimaging and dark and photodynamic cytotoxicity of silica nanoparticles doped by {Mo6I8}4+ metal clusters.

Silica nanoparticles (SNPs) doped by hexanuclear molybdenum cluster complexes [{Mo6X8}L6]n (X = Cl, Br, or I; L = various inorganic or organic ligands) have been recently suggested as materials with high potential for biomedical applications due to both their outstanding photoluminescence properties and their ability to efficiently generate singlet oxygen upon photoirradiation. However, no studies were undertaken so far to prove this concept. Therefore, here we examined the potential of photoluminescent SNPs doped by {Mo6I8}4+ for applications such as bioimaging and photodynamic therapy using the human epidermoid larynx carcinoma (Hep-2) cell line as a model. Our results demonstrated both: (i) significant luminescence from cells with internalised molybdenum cluster-doped SNPs combined with the low cytotoxicity of particles in the darkness and (ii) significant cytotoxicity of the particles upon photoirradiation. Thus, this research provides strong experimental evidence for high potential of molybdenum-cluster-doped materials in biomedical applications such as optical bioimaging, biolabeling and photodynamic therapy.

Consumer acceptability of liquorice root (Glycyrrhiza glabra L.) as an alternative sweetener and correlation with its bioactive content and biological activity

Consumer acceptability and sensory properties of liquorice root (Glycyrrhiza glabra L.) were evaluated. Quantitation of total polyphenolics and glycyrrhizic acid (GA), as well as the antioxidant capacity of liquorice extracts, was conducted and their biological effects (cytotoxic, antioxidative/pro-oxidative activity, lipid peroxidation on human laryngeal carcinoma cell line) compared to the ones of their predominant bioactive compound – GA. Conducted consumer survey revealed poor familiarity with liquorice (12.37% of correspondents), but willingness towards its use as an alternative sweetener (77.32% of consumers). Polyphenolic content of evaluated extracts ranged from 1018.18 to 1277.27 mg gallic acid equivalents (GAE)/l while GA content varied between 2179.53 and 2944.13 mg/l. The most pronounced cytotoxic effect (60%) and lipid peroxidation were exerted by treatment with the highest applied extract concentrations (10 mg/ml). Pure GA exhibited cytotoxic and pro-oxidative effects at concentrations of 0.12–0.6 mg/ml. Due to high GA content, coupled with its pronounced cytotoxic activity, the intake of liquorice root should be limited.

Comparative evaluation of PLGA nanoparticle delivery system for 5-fluorouracil and curcumin on squamous cell carcinoma

PurposeThe purpose of this study is to assess the effect of 5-fluorouracil nanoparticles and curcumin naoparticles on cell proliferation and the expression of the apoptotic marker (caspase 3) in squamous cell carcinoma cell line. Material and methodsPLGA 5-fluorouracil nanopartciles and PLGA curcumin nanoparticles were prepared and applied for 24 and 48h on human laryngeal squamous carcinoma cell line (Hep-2) as regard IC 50 concentration. MTT assay was used for evaluation of cytotoxicity of prepared nanoparticles. Quantitaive reverse transcriptase polymerase chain reaction (QRT-PCR) was used for the assessment of caspase-3 expression in the treated cell line. ResultsThe drug release rate profiles was dependent upon polymer to drug ratio, noting that the higher PLGA polymer ratio to 5-fluprouracil or curcumin drug showed faster release rates. On the other hand, the least PLGA polymer ratio to 5-fluprouracil or curcumin drug showed the slowest release rates. MTT assay revelaed that 5-fluorouracil nanoparticels or curcumin nanoparticels showed a clear cytotoxic effect on Hep-2 cell line compared to non treated cancer cells. The RT-PCR assessment of caspase-3 expression revealed that there was a significant increase in caspase-3 expression in Hep-2 cell line treated with 5-fluorouracil nanoparticles or curcumin compared to non treated cancer cells. ConclusionCurcumin nanoparticles could be more active in inducing apoptosis in short term assays (24h) than long term assays (48h) due to differential cellular uptake. While 5-fluorouracil nanoparticles induced higher significant apoptosis in long term (48h) compared to curcumin group.

Biogenic synthesis, characterization of antibacterial silver nanoparticles and its cell cytotoxicity

The advanced research and development of silver nanoparticles (AgNPs) is vast due to their incredible applications today. In this work, AgNPs were synthesized using soil derived Pseudomonas putida MVP2. The AgNPs formation on the P. putida cell membrane and its cell free supernatant was studied. The synthesized AgNPs were characterized by UV–visible spectroscopy, scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), energy dispersive X-ray (EDAX) and Fourier transform infrared (FTIR) spectrum analysis. The mode of action of AgNPs on the bacteria was studied against clinically isolated bacterial pathogens, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Helicobacter pylori by membrane integrity, and protein leakage using confocal and electron microscopy. Interestingly, AgNPs had no cytotoxicity under 25μg/mL and it was toxic at above 50μg/mL on human epidermoid larynx carcinoma (HEp-2) cells. This study evidenced that biogenic nanoparticles could affect the bacterial replication, protein leakage and eventually cell death. This might be used for active antimicrobial agents for the chronic infections.

The anti-tumour activity of rLj-RGD4, an RGD toxin protein from Lampetra japonica, on human laryngeal squamous carcinoma Hep-2 cells in nude mice.

The objective of this study is to investigate the antiproliferative activity and mechanism of integrin-binding rLj-RGD4 in a Hep-2 human laryngeal carcinoma-bearing nude mouse model. Human laryngeal squamous carcinoma cells (Hep-2) were inoculated subcutaneously into the axilla of nude mice to generate a Hep-2 human laryngeal carcinoma-bearing nude mouse model. When the Hep-2 xenograft model was successfully established, the animals were randomly separated into five groups. Three groups were treated with different dosages of rLj-RGD4. Cisplatin was administered to the positive control group, and normal saline (NaCl) was administered to the negative control group for 3 weeks. The body weights and the survival of the nude mice were evaluated, and the volumes and weights of the solid tumours were measured. The mechanism underlying rLj-RGD4 inhibition of tumour growth in transplanted Hep-2 human laryngeal carcinoma-bearing nude mice was evaluated by haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), measurement of intratumoural microvessel density (MVD), Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The tumour volumes and weights of the treatment groups were reduced compared with the model group, and survival times were improved by rLj-RGD4 treatment in Hep-2 human laryngeal carcinoma-bearing nude mice. The number of apoptotic Hep-2 human cells and intratumoural MVD significantly decreased after the administration of rLj-RGD4. In the xenograft tissue of animals treated with rLj-RGD4, FAK, PI3K, and Akt expression was unaltered, whereas P-FAK, P-PI3K, Bcl-2, P-Akt, and VEGF levels were down-regulated. In addition, activated caspase-3, activated caspase-9, and Bax levels were up-regulated. rLj-RGD4 exhibits potent invivo activity and inhibits the growth of transplanted Hep-2 human laryngeal carcinoma cells in a nude mouse model. Thus, these results indicate that the recombinant RGD toxin protein rLj-RGD4 may serve as a potent clinical therapy for human laryngeal squamous carcinoma.

Haemophilus influenzae triggers autophagy in HEp-2 cells.

The MAP-LC3 system regulates the intracellular formation of autophagy-associated vacuoles. These vacuoles contain the LC3 protein; thus it has been utilized as a marker to identify autophagosomes. The aim of our study was to investigate whether Haemophilus influenzae strains and their supernatants could activate autophagy in human larynx carcinoma cell line (HEp-2). We demonstrate that higher expression of the LC3B-II protein was induced, particularly by nontypeable Haemophilus influenzae (NTHi) 49766 and by supernatants, containing <50 kDa proteins, of both strains. Ultrastructural studies demonstrate vacuoles with a double membrane and/or membrane material inside, showing similar features to those of autophagic vacuoles. Together, our findings demonstrate that H. influenzae strains and their supernatants trigger an autophagic process.