Human Laryngeal Carcinoma Hep-2 Cells Research Articles

Polyvinyl Alcohol Capped Silver Nanostructures for Fortified Apoptotic Potential Against Human Laryngeal Carcinoma Cells Hep-2 Using Extremely-Low Frequency Electromagnetic Field.

: Polyvinyl alcohol-capped silver nanostructures (cAgNSs) were investigated in order to enhance the cytotoxicity, pro-apoptotic, and oxidant patterns of in human laryngeal carcinoma Hep-2 cells by employing a 50 mT electromagnetic field (LEMF) for 30 min. Wet chemical reduction was used to synthesize the cAgNSs, and after they had been capped with polyvinyl alcohol, they were specifically examined for particle size analysis and structural morphology. To visualize how the silver may attach to the protein targets, a molecular docking study was conducted. Estimation of cytotoxicity, cell cycle progression supported by mRNA expression of three apoptotic-promoting genes and one apoptotic-resisting. Particle size analysis results were a mean particle size of 157.3±0.5 nm, zeta potential value of -29.6 mV±1.5 mV, and polydispersity index of 0.31±0.05. Significantly reduction of IC50 against Hep-2 cells by around 6-fold was concluded. Also, we obtained suppression of the proliferation of Hep-2 cells, especially in the G0/G1 and S phases. Significant enhanced mRNA expression revealed enhanced induced CASP3, p53, and Beclin-1 mediated pro-apoptosis and induced NF-κB mediated autophagy in Hep-2 cells. Augmented levels of GR, ROS and MDA as oxidative stress biomarkers were also obtained. HE staining of Hep-2 cells exposed to cAgNSs and LEMF confirmed the enhanced apoptotic potential comparatively. By conclusion, the developed nano-sized structures with the aid of extremely-low frequency electromagnetic field were successful to fortify the anti-cancer profile of cAgNSs in Hep-2 cells.

Citalopram, an antipsychotic agent, induces G1/G0 phase cell cycle arrest and promotes apoptosis in human laryngeal carcinoma HEP-2 cells

Human laryngeal squamous carcinoma (LSCC) is a common malignant tumor in the head and neck. Despite the recently developed therapies for the treatment of LSCC, patients’ overall survival rate still did not enhance remarkably; this highlights the need to formulate alternative strategies to develop novel treatments. The antitumor effects of antidepressant drugs such as citalopram have been reported on several cancer cells; however, they have yet to be investigated against LSCC. The current study was directed to explore the possible antitumor effects of citalopram on human laryngeal carcinoma cell lines (HEP-2). HEP-2 cells were cultured and treated with different doses of citalopram (50–400 µM) for 24, 48, and 72 h. The effects of citalopram on the viability of cancer cells were determined by the MTT assay. In addition, apoptosis and cell cycle analysis were performed by flow cytometry. Moreover, evaluation of the expression of proapoptotic and apoptotic proteins, such as cytochrome c, cleaved caspases 3 and 9, Bcl-2, and BAX, was performed by western blotting analysis. Our results revealed that citalopram significantly suppressed the proliferation of HEP-2 cells through the upregulation of p21 expression, resulting in the subsequent arrest of the cell cycle at the G0/G1 phase. Furthermore, citalopram treatment-induced HEP-2 cell apoptosis; this was indicated by the significant increase of cytochrome c, cleaved caspases 3 and 9, and BAX protein expression. On the contrary, Bcl-2 protein expression was significantly downregulated following treatment with citalopram. The ultrastructure studies were in accordance with the protein expression findings and showed clear signs of apoptosis with ring chromatin condensation upon treatment with citalopram. These findings suggest that citalopram’s anti-tumor activities on HEP-2 cells entailed stimulation of the intrinsic apoptotic pathway, which was mediated via Bcl-2 suppression.

Oridonin inhibited epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling.

Oridonin is the main bioactive component of Rabdosia rubescens, and its anticancer activity has been reported in a variety of cancers. However, the molecular mechanism of oridonin in laryngeal carcinoma remains unclear. In the present study, the cytotoxic effect of oridonin on laryngeal carcinoma Hep-2 and TU212 cell lines were initially detected by modified MTT assay. The results showed that oridonin had a dose-dependent anti-proliferative effect on laryngeal carcinoma Hep-2 and TU212 cells. Next, we found that oridonin significantly inhibited the migration and invasion of human laryngeal carcinoma Hep-2 and TU212 cell lines by wound healing assay and transwell assay. Subsequently, the results of quantitative real-time PCR assay and western blotting assay confirmed that oridonin upregulated the expression of E-cadherin while downregulated the expression of N-cadherin in a concentration-dependent manner at mRNA and protein levels. In addition, phosphorylation levels of liver kinase B1 (p-LKB1) and AMP-activated protein kinase (p-AMPK) were also elevated upon oridonin treatment. To further verify the role of LKB1/AMPK signaling pathway in laryngeal carcinoma, overexpression of LKB1 was constructed by plasmid transfection. The data exhibited that overexpression of LKB1 could further reinforce the increase of E-cadherin level and decrease of N-cadherin level mediated by oridonin. Additionally, AMPK inhibitor compound C could reverse anti-metastatic effect of oridonin on laryngeal carcinoma, and antagonise EMT expression. In contrast, AMPK activator AICAR presented the opposite effect. In conclusion, our study revealed that oridonin could remarkably reverse the epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling pathway, which suggested that oridonin may be a potential candidate for the treatment of laryngeal carcinoma in the future.

Magnetic Double Hydrogen Artesunate Nano Liposome on CAL Tongue Squamous Carcinoma and Laryngeal Hep-2-27 Cell Lines: An In Vitro Study

With the continuous development of nanomedicine, scholars have reported many studies on the application of nanocarriers to improve the efficiency of tumor diagnosis and treatment. We designed magnetic nanoparticles combined with liposomes to carry DHA (DHA-MAG-NL) nanoparticles to improve the biocompatibility of DHA, protect DHA activity, and increase DHA blood concentration. To study the inhibitory effect of magnetic dihydroartemisinin nano-liposomes on the proliferation of human tongue squamous cell carcinoma CAL-27 cell line and laryngeal carcinoma Hep-2 cell line In Vitro. The effect of DHA-Mag-NL concentration gradient on the proliferation of Hep-2 and Cal-27 cells at different intervention times was detected by MTT assay. The effects of DHA concentration of 40 μM on the cell cycle and apoptosis of Hep-2 and Cal-27 were analyzed by flow cytometry. Statistical analysis showed that DHA-Mag-NL had a weak inhibitory effect on Cal-27 cells. The apoptosis-promoting effect of DHA-Mag-NL on Cal-27 tumor cells was weaker than that of DHA for 24 h. We concluded that DHA-Mag-NL can inhibit the proliferation of Hep-2 and Cal-27 tumor cells and promote apoptosis, which is dependent on drug dose and intervention time.

КЛИНИКО-ДИАГНОСТИЧЕСКОЕ ЗНАЧЕНИЕ АНТИНУКЛЕАРНЫХ АНТИТЕЛ В ПРАКТИКЕ ПЕДИАТРА (ЧАСТЬ I)

In recent decades, there has been an increase in the incidence of various autoimmune, including rheumatic diseases, while their debut has been shifted to an earlier age. The clinical picture of rheumatic diseases is distinguished by severe polymorphism; therefore, laboratory diagnostic methods are important for diagnostic search. One of the most widely used tests in the rheumatological practice is the detection and identification of antinuclear antibodies (ANA). ANA are a large group of autoantibodies (auto-Abs) directed against various cell structures, including not only the nucleus but also the nuclear membrane, mitotic apparatus, cytoplasm components and cell organelles, as well as cell membranes. Different methods are used for testing, but the method of ANA detection through indirect immunofluorescence antinuclear antibody test (IF-ANA) is recommended as a “gold standard” with human laryngeal carcinoma HEp-2 cells being recognized as a standard substrate. IF-ANA with HEp-2 has the highest diagnostic sensitivity compared with other methods, it includes the phase of serial dilutions of positive serums and a visual analysis of the type of glow, the list of which currently has 30 patterns. ANA glow types are caused by various auto-Abs that respond with antigens located in the nucleus and cytoplasm of the HEp-2 cells line. Assessment of the type of glow in conjunction with clinical and laboratory data allows planning of further examination with confirming tests to determine the specificity of ANA, some of which, in particular, are auto-Abs to DFS70 antigen, which in the absence of other types of autoantibodies, are more likely to doubt the presence of a systemic rheumatic disease. The diagnostic specificity of various screening techniques for ANA detection is relatively low, they are detected with a fairly high frequency in patients with other diseases and in healthy individuals of different gender and age, including children.

Synthetic curcumin analog: inhibiting the invasion, angiogenesis, and metastasis in human laryngeal carcinoma cells via NF-kB pathway.

Laryngeal carcinoma, the most common among head and neck squamous cell carcinoma (HNSCC), induces 1% of all cancer deaths. Curcumin the active constituent of turmeric, is shown to be effective in the treatment of various cancers. In the present study, we explored the mechanistic role of bis-demethoxy curcumin analog (BDMC-A) as a chemotherapeutic agent. We investigated its inhibitory effect on invasion, angiogenesis, and metastasis in human laryngeal carcinoma (Hep-2) cells in comparison with curcumin. The effect of curcumin and BDMC-A on transcription factors (NF-κB, p65, c-Jun, c-Fos, STAT3, 5, PPAR-γ, β-catenin, COX-2, MMP-9, VEGF, TIMP-2) involved in signal transduction cascade, invasion, and angiogenesis in Hep-2 cells were quantified using Western blotting and RT-PCR technique. ELISA was used to measure the pro-inflammatory markers in Hep-2 cells treated with curcumin and BDMC-A. The results showed that BDMC-A inhibits the transcription factors NF-κB, p65, c-Jun, c-Fos, STAT3, STAT5, PPAR-γ and β-catenin, which are responsible for tumor progression and malignancy. Moreover, BDMC-A treatment downregulated MMP-9, VEGF, TGF- β, IL-6 and IL-8 and upregulated TIMP-2 levels. The effects were more significant compared to curcumin. Our overall results revealed that BDMC-A more effectively inhibited the markers of invasion, angiogenesis and metastasis in comparison with curcumin.

The therapeutic effects of Ficus carica extract as antioxidant and anticancer agent

Natural extracted ingredients are evolutionarily selected molecules and are used to control inflammation, and cancerous progression and transformation. Several studies have evaluated the beneficial properties of certain pharmaceutical molecules extracted from Ficus carica (FC). The current study aimed to investigate the antioxidant and anticancer properties of FC ethanolic leaves extract. The dried coarse powder of F. carica leaves was exhaustively extracted with ethanol. The resulting crude ethanolic F. carica extract (FCE) was assayed for total phenolic content and, antioxidant and anticancer activities, since both are interrelated. The antioxidant activity of the FCE was determined using DPPH as a standard stable free radical. The anticancer activity of the extract was determined against breast cancer (MCF7), hepatocellular carcinoma (HepG2), Colon cancer (CaCo-2), and a human laryngeal carcinoma (Hep-2) cell lines. The obtained results indicated a strong antioxidant activity of the extract with 1 mg/ml having 75.7% DPPH scavenging ability. The anti-cancer activities of the extract showed strong inhibition percentages against all the selected cell lines. The FCE had potent effects against both Hep2 and HepG2 cells with inhibition percentages ranged 80.7–66.9%. The properties of the FCE demonstrated here support our proposal of the validity of the traditional health claims of medicinal plants.

Abstract P-39: Fluorescent Silver Nanoclusters with Immunoglobulins and Albumins

Background: Multiplex biomedical assays including molecular genetic tests and immunoanalysis require multiple fluorophores with a wide excitation range and different emission spectra. In comparison with organic fluorophores and quantum dots, the metal nanoclusters (NC) consisting of a few to hundred atoms have the following advantages: small size, large Stokes shift, prolonged fluorescence lifetime and biocompatibility. Our research was aimed at construction of fluorescent AgNC with the main blood proteins and transmission electron microscopy (TEM). Methods: AgNC were synthesized from AgNO3 in the presence of albumins and immunoglobulins (Ig) of different classes and origin at pH 9-11 with NaBH4 recovery. The resulting AgNC with proteins were loaded to "Formvar/Carbon 200 Mesh Copper" copper grids (Ted Pella, USA) and examined using TEM system JEM 2100 Plus (JEOL, Japan) without contrast. Fluorescence excitation/emission spectra were measured in quartz cuvette using the FluoroMax + spectrofluorometer (Horiba Scientific, Japan). Results: Recovery of Ag+ ions did not occur in the presence of IgG and albumins without NaBH4 at different temperatures, pH, and incubation time. Broad excitation spectra of AgNC were in a range 340-540 nm. Their emission spectra correlated with the original AgNO3 concentration and did not depend on protein and pH. NC stabilized with IgG or albumin with blue fluorescence and emission maximum at 420 nm contained NC from 0.6 nm and higher. Green AgNC with proteins had bright fluorescence at 430-470 nm and red NC showed emission maximum at 650 nm. TEM revealed discrete AgNC and their numerous aggregates in each sample of fluorescent NC in spite of different fluorescent emission spectra. According to the MTT test, AgNC with human IgG and BSA with protein concentrations up to 3 mg/ml were not toxic for human larynx carcinoma HEp-2 cells despite cytotoxicity of silver nanoparticles covered with IgG or albumin envelopes as well as Cd and AuNC with BSA. Conclusion: AgNC with antibodies and albumin with a broad size range and aggregation possess tunable fluorescence emission spectra with broad excitation at 340-540 nm. Different emission spectra permit AgNC to be used in multiplex assays. AgNC were not toxic for human tissue culture and may be applied for bioimaging.

PH-Sensitive Dairy-Derived Hydrogels with a Prolonged Drug Release Profile for Cancer Treatment.

A novel versatile biocompatible hydrogel of whey protein isolate (WPI) and two types of tannic acid (TAs) was prepared by crosslinking of WPI with TAs in a one-step method at high temperature for 30 min. WPI is one common protein-based preparation which is used for hydrogel formation. The obtained WPI-TA hydrogels were in disc form and retained their integrity after sterilization by autoclaving. Two TA preparations of differing molecular weight and chemical structure were compared, namely a polygalloyl glucose-rich extract-ALSOK 02-and a polygalloyl quinic acid-rich extract-ALSOK 04. Hydrogel formation was observed for WPI solutions containing both preparations. The swelling characteristics of hydrogels were investigated at room temperature at different pH values, namely 5, 7, and 9. The swelling ability of hydrogels was independent of the chemical structure of the added TAs. A trend of decrease of mass increase (MI) in hydrogels was observed with an increase in the TA/WPI ratio compared to the control WPI hydrogel without TA. This dependence (a MI decrease-TA/WPI ratio) was observed for hydrogels with different types of TA both in neutral and acidic conditions (pH 5.7). Under alkaline conditions (pH 9), negative values of swelling were observed for all hydrogels with a high content of TAs and were accompanied by a significant release of TAs from the hydrogel network. Our studies have shown that the release of TA from hydrogels containing ALSOK04 is higher than from hydrogels containing ALSOK 02. Moreover, the addition of TAs, which display a strong anti-cancer effect, increases the cytotoxicity of WPI-TAs hydrogels against the Hep-2 human laryngeal squamous carcinoma (Hep-2 cells) cell line. Thus, WPI-TA hydrogels with prolonged drug release properties and cytotoxicity effect can be used as anti-cancer scaffolds.

L-ascorbic acid induces apoptosis in human laryngeal epidermoid Hep-2 cells by modulating the nuclear factor kappa-light-chain-enhancer of activated B cells/ mitogen-activated protein kinase/Akt signaling pathway

L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl- 2 family and MAPK/Akt signaling pathways.

Magnesium Isoglycyrrhizinate Induces an Inhibitory Effect on Progression and Epithelial-Mesenchymal Transition of Laryngeal Cancer via the NF-κB/Twist Signaling.

BackgroundMagnesium isoglycyrrhizinate (MI) was extracted from roots of the plant Glycyrrhiza glabra, which displays multiple pharmacological activities such as anti-inflammation, anti-apoptosis, and anti-tumor. Here, we aimed to investigate the effect of MI on the progression and epithelial–mesenchymal transition (EMT) of laryngeal cancer.MethodsForty laryngeal cancer clinical samples were used. The role of MI in the proliferation of laryngeal cancer cells was assessed by MTT assay, Edu assay and colony formation assay. The function of MI in the migration and invasion of laryngeal cancer cells was tested by transwell assays. The effect of MI on apoptosis of laryngeal cancer cells was determined by cell apoptosis assay. The impact of MI on tumor growth in vivo was analyzed by tumorigenicity analysis using Balb/c nude mice. qPCR and Western blot analysis were performed to measure the expression levels of gene and protein, respectively.ResultsWe identified that EMT-related transcription factor Twist was significantly elevated in the laryngeal cancer tissues. The expression of Twist was also enhanced in the human laryngeal carcinoma HEP-2 cells compared with that in the primary laryngeal epithelial cells. The high expression of Twist was remarkably correlated with poor overall survival of patients with laryngeal cancer. Meanwhile, our data revealed that MI reduced cell proliferation, migration and invasion and enhanced apoptosis of laryngeal cancer cells in vitro. Moreover, MI decreased transcriptional activation and the expression levels of NF-κB and Twist, and alleviated EMT in vitro and in vivo. MI remarkably inhibited tumor growth and EMT of laryngeal cancer cells in vivo.ConclusionMI restrains the progression of laryngeal cancer and induces an inhibitory effect on EMT in laryngeal cancer by modulating the NF-κB/Twist signaling. Our finding provides new insights into the mechanism by which MI inhibits laryngeal carcinoma development, enriching the understanding of the anti-tumor function of MI.

A Raman Spectroscopic Study of Thymoquinone Antitumor Action

Fluorescence assay and Raman spectroscopy were used to study the mechanisms of action of 2-isopropyl-5-methyl- 1,4-benzoquinone (thymoquinone) on HEp-2 human larynx carcinoma cells. Thymoquinone was found to have a more pronounced toxic effect than 1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone. The action of thymoquinone leads to a decrease in the mitochondrial membrane potential and release of cytochrome c from the mitochondria, indicating activation of apoptosis of tumor cells through the mitochondrial-mediated pathway. These results indicate the possibility of using Raman spectroscopy in the study of programmed cell death.

Study on the mechanism of inhibiting invasion of human laryngeal squamous cell carcinoma Hep-2 and TU212 cells after the downregulation of miRNA-106b

Objective To investigate the effect of miRNA-106b (miR-106b) on human laryngeal squamous cell carcinoma Hep-2 and TU212 cells and its mechanism. Methods Hep-2 and TU212 cells were divided into miR-106b inhibitory sequence transfected group (the experimental group), miR-106b competitive negative sequence transfected group (the negative control group) and non-intervention group (the blank group). The inhibitory effect of miR-106b inhibitory sequence on the expression of miR-106b was verified by using reverse transcription quantitative polymerase chain reaction (qRT-PCR). Whether phosphatase and tensin homolog (PTEN) was the target gene of miR-106b was analyzed by using bioinformatics and luciferase report vector. PTEN small interfering RNA (siRNA) was used to inhibit the expression of PTEN in Hep-2 and TU212 cells. Transwell method and Western blot were used to detect the change of invasion ability of Hep-2 and TU212 cells after miR-106b silencing or the PTEN intervening, and the expression change of PTEN, epithelial cadherin and vimentin. Results The relative expression levels of miR-106b in Hep-2 and TU212 cells in the experimental group were 0.110 ± 0.037 and 0.074 ± 0.009, respectively, which were lower than those in the negative control group (1.013±0.059 and 1.035±0.062, respectively; all P < 0.05). In Transwell experiments, the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group was less than that in the negative control group [(37.09±4.02) vs. (95.65±4.77), (29.16±2.49) vs. (103.19±6.08), all P < 0.05]. The bioinformatics analysis results showed that 3'-UTR region of PTEN mRNA was complementary to 3'-UTR region of miR-106b. Dual-luciferase reporter system analysis showed that the luciferase reporter activity of wild-type PTEN gene transfected with miR-106b was decreased to (22.84±2.68)%, and that of mutant PTEN gene transfected with miR-106b was almost unchanged [(92.08±3.44)%], and the difference was statistically significant (P < 0.001). The expression level of PTEN protein of Hep-2 and TU212 cells in the experimental group was higher than that in the negative control group. Transwell method showed that the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression was more than that in the experimental group without the inhibition of PTEN expression [(65.08±3.57) vs. (26.72±2.58), (57.38±4.96) vs. (31.81±2.97), all P < 0.05]. Western blot showed that the expression level of epithelial-cadherin was up-regulated and vimentin was down-regulated of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression. Conclusions The human laryngeal squamous cell carcinoma Hep-2 and TU212 cell miR-106b can influence the downstream invasion-related protein of PTEN and change the cell invasion ability through the targeted regulation of PTEN expression. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; RNA interference; miRNA-106b; Phosphatase and tensin homolog

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells.

Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. MTT assay was used to detect the inhibitory rate of viability. Giemsa's staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. These results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

Cytotoxic activity and volatile components of peel oil of Citrus volkameriana

Citrus volkameriana Pasq. (Family: Rutacea) is a very common edible and medicinal plant growing in several places in the world including Egypt. We previously studied the toxicity of components of the leaves; however, the peel oil has not been fully analyzed. In the current study, peel oil of C. volkameriana was prepared by hydro-distillation method, and was analyzed for its components using gas chromatography and gas chromatography–mass spectrometry. The recovered oil was formulated as oil in water emulsion using a wet gum technique with gum acacia as an emulsifier. Two compounds were selected from the identified oil components, emulsified using the same technique, and tested for their cytotoxicity along with the emulsified oil on five human tumor cell lines, as well as on one human non-tumorigenic epithelial breast cell line. Our results showed that limonene (68.5%) was found to be the major oil constituent, followed by γ-terpinene (11.3%). Other minor oil components include thymol methyl ether (4%), 4-terpineol (2.9%), cymol (2.4%) and α-pinene (2.1%). The oil showed a very strong cytotoxic effect on the five human tumor cells tested with the highest effect on human hepatoma HepG2 cells (IC50 = 0.038 μL/mL), followed by the effect on human laryngeal carcinoma HEp2 cells (IC50 = 0.045 μL/mL), human breast adenocarcinoma MCF7 cells (IC50 = 0.075 μL/mL), human lung carcinoma A549 cells (IC50 = 0.093 μL/mL), and human cervical cancer Hela cells (IC50 = 0.13 μL/mL). In contrast, the oil showed a lower cytotoxic effect on the non-tumorigenic breast MCF10A cells (IC50 = 0.106 μL/mL). D-limonene possessed similar cytotoxic results as the oil, suggesting a vital role in the cytotoxic effect obtained with the oil. Furthermore, α-pinene showed a strong cytotoxic effect on MCF7 cells (IC50 = 0.072 μL/mL) with a weaker effect on the MCF10A cells (IC50 = 0.185 μL/mL). In conclusion, Citrus volkameriana peel oil and its components have the potential to act as promising cytotoxic agents.

Mechanisms of IFNalpha-1a-Induced Apoptosis in a Laryngeal Cancer Cell Line.

BackgroundInterferon alpha (IFNalpha) exerts its anti-proliferative effect on many human cancers. Among the 13 subtypes of human IFNalpha, IFNalpha-1 subtype has 2 variants, named IFNalpha-1a and IFNalpha-1b, that differ from each other in only 1 amino acid, at residue 114. However, the mechanism by which IFNalpha-1a mediates growth inhibition is still unclear.Material/MethodsHuman laryngeal carcinoma HEp2 cells were treated with IFNalpha-1a by either transient transfection or exogenous delivery. Western blot and RT-PCR analysis were carried out to assess apoptotic pathways active in IFNalpha-1a-treated HEp2 cells. Microarray analysis was conducted to uncover the differential gene expressions after IFNalpha-1a treatment. KEGG pathway enrichment analysis was also performed.ResultsIFNalpha-1a markedly inhibited the proliferation and significantly promoted the apoptosis of HEp-2 cells. Mechanistic studies indicate that IFNalpha-1a-mediated cell apoptosis is directly linked to intrinsic and endoplasmic reticulum (ER) stress-related apoptosis, but is independent of extrinsic apoptosis. The top 40 differentially expressed genes discovered by microarray analysis included 20 upregulated genes (e.g., IFI6, IFI27, IFI44L, and MIR548X) and 20 downregulated genes (e.g., PRKDC, HIST1H3B, DYNC1H1, and HIST1H2AM). KEGG pathway enrichment analysis revealed that 4 out of 6 pathways are TP53-related.ConclusionsWe demonstrated a detailed mechanism involved in IFNalpha-1a-mediated anti-proliferation activity in human laryngeal carcinoma cells.

Knockdown of YAP inhibits growth in Hep-2 laryngeal cancer cells via epithelial-mesenchymal transition and the Wnt/\u03b2-catenin pathway

BackgroundYes-associated protein (YAP) plays a crucial role in tumour development and it is the main effector of the Hippo signalling pathway. However, the mechanism underlying YAP downregulation in laryngeal cancer is still unclear. In our previous study, we found that YAP, compared with adjacent tissues, was expressed higher in laryngeal cancer and was also closely associated with histological differentiation, TNM stage and poor prognosis.MethodsIn this study, we attempted to determine whether silenced YAP could downregulate human laryngeal carcinoma Hep-2 cells progression. YAP was downregulated in Hep-2 cells by shRNA, and the malignant ability of Hep-2 was assessed in vitro and in vivo.ResultsIn vitro, CCK-8, colony formation and wound healing assays showed that downregulation of YAP significantly reduced the rates of proliferation, migration, and invasion in Hep-2 cells. Downregulation of YAP distinctly induced G2/M cycle arrest and increased the rate of apoptosis. Accordingly, western blot assay suggested that the expression of DKK1, vimentin and β-catenin was significantly decreased after YAP downregulated treatment, thereby indicating that YAP mediated the EMT programme and the Wnt/β-catenin signalling pathway in carcinoma of the larynx. Furthermore, silencing of YAP suppressed Hep-2 cell tumourigenesis and metastasis in vivo.ConclusionIn summary, our findings demonstrated the proliferation of YAP downregulation and the invasion of Hep-2 cells via downregulating the Wnt/β-catenin pathway in vitro and in vivo, suggesting that YAP may provide a potential therapeutic strategy for the treatment of laryngeal cancer.

Partial characterization and antitumor activity of a polysaccharide isolated from watermelon rinds

As a health-beneficial fruit, watermelon is widely consumed by people around the world. However, components responsible for the health benefits are not yet determined. As watermelon contains a large amount of polysaccharides, these carbohydrates might play an important role in the health benefits. In this work, polysaccharide from watermelon rinds (PWR) was extracted by papain digestion, purified and characterized by GC–MS, SEC/MALS/VD/DRI, FTIR and 1D and 2D NMR which revealed the glycosidic linkages, their locations in branches and backbone. The monosaccharide composition revealed that the extracted polysaccharide was composed of galactose (38.26%), arabinose (26.12%), rhamnose (17.86%), mannose (9.94%), xylose (5.10%) and glucose (2.70%) with a percentage of uronic acid of 45%. A combination of CPG and NMR analysis showed that the extracted polysaccharide is arabinogalactan linked to type I rhamnogalacturonan. we notice that the arabinogalactan was formed by →6)-β-D-Galp-(1→ as backbone with short branching of arabinose linked in α 1 → 3, rhamnose linked in α 1 → 4, mannose linked in β 1 → 6 and galactose branches linked in β 1 → 3. Furthermore, PWR exhibited obvious cytotoxicity ability to human laryngeal carcinoma Hep-2 cells in a dose-and time-dependant manner.

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

Long chain non-coding RNA MALAT-1 gene knockdown inhibits growth and migration and promotes apoptosis of human laryngeal squamous cell carcinoma Hep-2 cells in vitro

To investigate the effect of knocking down long chain non-coding RNA MALAT-1 gene on the biologicalbehaviors of human laryngeal squamous cell carcinoma Hep-2 cells. With immortalized nasopharyngeal epithelial(NPE) cell line NP-69 as the reference, MALAT1 expression in FaDu, Hep-2 and nasopharyngeal carcinoma CNE-2Z cells weredetected using real-time PCR. Hep-2 cells were transfected with shmalat1 lentivirus and the expression of MALAT1 wasdetected. MTT assay, flow cytometry, Transwell assay and M Atrigel invasiveness test were used to evaluate the effect ofMALAT-1 knockdown on the proliferation, cell cycle, cell apoptosis, migration, and invasiveness of Hep-2 cells. Compared with NP-69 cells, Hep-2 cells, FaDu cells, and CNE-2Z cells all showed significantly increased MALAT-1expression. In Hep-2 cells, knockdown of MALAT-1 significantly inhibited the cell proliferation, increased the cell percentagein S phase (P < 0.01), decreased the cell percentage in G2/M phase (P < 0.01), and attenuated the migration and invasiveness of thecells. MALAT-1 is over-expressed in laryngeal squamous cell carcinoma, and knocking down MALAT-1 gene cansignificantly suppress the proliferation, invasion and migration and promotes apoptosis of the cancer cells.