Human Laryngeal Cancer Cell Line Research Articles

Bioinformatic analysis reveals prognostic value and immunotherapy potential of Siglec-15 in laryngeal squamous cell carcinoma

BackgroundLaryngeal squamous cell carcinoma (LSCC) is the ultimate common malignant head and neck cancer with dismal prognosis. The expression pattern and clinical significance of Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) in LSCC are poorly understood. In order to lay the groundwork for future immune-related research on Siglec-15 in LSCC, we set out to study its expression and prognostic importance in the disease, as well as to use bioinformatics to investigate the immune features modulated by Siglec-15 in LSCC. Methods① In order to get the gene expression profile and clinical data for TCGA head and neck cancer (TCGA-HNSC), you may access the relevant data from UCSC xena and use 110 cases of laryngeal cancer as a training set. Two datasets, GSE27020 and GSE25727, were obtained from the GEO databank and utilized as validation sets. These datasets include expression profiles and clinical information. The Siglec-15 gene and immune characteristics were analyzed by bioinformatics methods. ② Retrospectively collected routine paraffin specimens from patients with pathological diagnosis of squamous cell carcinoma from December 2012 to November 2015 in Sun Yat-sen Memorial Hospital and fresh frozen tissue of patients from June 2021 to March 2022. Immunohistochemistry method, immunofluorescence technique and real-time quantitative PCR was used to examine the difference of Siglec-15 appearance in LSCC tissue and adjacent tissue, and its correlation of prognosis, clinic pathological characteristics and CD8+T lymphocyte infiltration. Using human laryngeal cancer cell line (LCC), we studied the influence of Siglec-15 in cell proliferation and invasion. ResultsWe identified Siglec-15 was upregulated in LSCC. The patients in Siglec-15 high expression group had a poor overall survival (OS) based on the clinical information from TGCA and 111 LSCC patients that hospitalized in Sun Yat-sen Memorial Hospital. The COX regression analysis indicated Siglec-15 as an independent predictor for poor prognosis of LSCC. Bioinformatic analysis suggested that the high expression of Siglec-15 shape an immune suppressive tumor microenvironment (TEM), leading to poor response to immunotherapy in LSCC. Siglec-15 enhanced cell invasion and proliferation, as we showed in vitro. ConclusionOur study support Siglec-15 as a potential predictor for LSCC prognosis and an attractive target for LSCC immunotherapy.

Anticancer Potential of the Leaf Extracts from the Selected Croton Species

The reported investigations were aimed at establishing anticancer potential of the leaf extracts from Croton dictyophlebodes (CDE), C. kilwae (CKE) and C. longipedicellatus ssp austrotanzanicus (CLE). In vitro cytotoxicity assays of the extracts were performed against human laryngeal cancer cell line (HEp-2) and human breast cancer cell line (MCF-7), while in vivo toxicological studies were done using Swiss albino mice (Mus musculus) following the Organization for Economic Cooperation and Development guideline number 423 of 2001. Results for in vitro assays revealed that CDE and CLE induced cytotoxicity to MCF-7 and HEp-2 cell lines at EC50 values of 86 and 63 µg/mL, respectively, while CKE was inactive to HEp-2 cells at 100 µg/mL. For in vivo studies, mice were given calculated doses based on globally harmonized standards and monitored for 14 days to observe signs of toxicity. Haematological analysis was also carried out to obtain information on changes in blood conditions. The findings showed that LD50 for CKE and CDE were > 2000 mg/kg, while for CLE was 175 mg/kg body weight (b.w.). The oral administration of CDE and CKE did not reveal significant changes in haematological parameters of treated mice, while CLE provoked some. Thus, the studied extracts may be considered potential cancer remedies with safety pre-cautions that warrant further investigations.

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression.

Percutaneous coronary intervention (PCI) is the most widely used therapy for treating ischemic heart disease. However, intimal hyperplasia and restenosis usually occur within months after angioplasty. Modern pharmacological researchers have proven that osthole, the major active coumarin of Cnidium monnieri (L.) Cusson, exerts potent antiproliferative effects in lung cancer cells, the human laryngeal cancer cell line RK33 and TE671 medulloblastoma cells, and its mechanism of action is related to cell cycle arrest. The goal of the present study was to observe the effect of osthole on vascular smooth muscle cell (VSMC) proliferation using platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs isolated from rats and vascular balloon injury as models to further elucidate the molecular mechanisms underlying this activity. We detected the relative number of VSMCs by the MTT assay and EdU staining and examined cell cycle progression by flow cytometry. To more deeply probe the mechanisms, the protein expression levels of PCNA, the cyclin D1/CDK4 complex and the cyclin E1/CDK2 complex in balloon-treated rat carotid arteries and the mRNA and protein expression levels of the cyclin D1/CDK4 and cyclin E1/CDK2 complexes in VSMCs were detected by real-time RT-PCR and western blotting. The data showed that osthole significantly inhibited the proliferation of VSMCs induced by PDGF-BB. Furthermore, osthole caused apparent VSMC cycle arrest early in G0/G1 phase and decreased the expression of cyclin D1/CDK4 and cyclin E1/CDK2. Our results demonstrate that osthole can significantly inhibit PDGF-BB-induced VSMC proliferation and that its regulatory effects on cell cycle progression and proliferation may be related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase. The abovementioned mechanism may be responsible for the alleviation of neointimal hyperplasia in balloon-induced arterial wall injury by osthole.

Histone Deacetylase Inhibitors Enhance Cell Killing and Block Interferon-Beta Synthesis Elicited by Infection with an Oncolytic Parainfluenza Virus.

Previous results have shown that infection with the cytoplasmic-replicating parainfluenza virus 5 mutant P/V-CPI- sensitizes cells to DNA damaging agents, resulting in the enhanced killing of airway cancer cells. Here, we have tested the hypothesis that histone deacetylase (HDAC) inhibitors can also act with P/V-CPI- infection to enhance cancer cell killing. Using human small cell lung cancer and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid-mediated enhanced killing was eliminated in lung cancer cells that were engineered to express a protein which sequesters double stranded RNA. Scriptaid also enhanced cancer cell killing by two other negative strand RNA viruses – the La Crosse virus and vesicular stomatitis virus. Scriptaid treatment enhanced the spread of the P/V-CPI- virus through a population of cancer cells, and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). Taken together, these data support a role for combinations of a cytoplasmic-replicating RNA virus such as the P/V-CPI- mutant along with chemotherapeutic agents.

In-silico and in-vitro anti-cancer potential of a curcumin analogue (1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-dione

PurposePreviously we showed that BDMC, an analogue of curcumin suppresses growth of human breast and laryngeal cancer cell line by causing apoptosis. Here, we demonstrate the enhanced anti-cancer activity of a heterocyclic ring (indole) incorporated curcumin analogue ((1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-Dione), ICA in short, in comparison to curcumin. MethodICA was synthesized by a one pot condensation reaction. Anti-cancer potential of ICA was assessed in three human cancer cell lines of different origin (Lung adenocarcinoma (A549), leukemia (K562) and colon cancer (SW480)) by MTT assay. Mode of cell death was determined by acridine orange-ethidium bromide (Ao-Eb) staining. Putative cellular targets of ICA were investigated by molecular docking studies. Cell cycle analysis following curcumin or ICA treatment in SW480 cell line was carried out by flow cytometry. Expression levels of Cyclin D1 and apoptotic markers, such as Caspase 3, 8 and 9 were studied by western blot analysis in SW480 cell line treated with or without ICA and curcumin. ResultsThe yield of ICA synthesis was found to be 69% with a purity of 98%. ICA demonstrated promising anti-cancer activity compared to curcumin alone, as discerned by MTT assay. ICA was non-toxic to the cell line of normal origin. We further observed that ICA is ∼2 fold more potent than curcumin in inhibiting the growth of SW480 cells. Ao-Eb staining revealed that ICA could induce apoptosis in all the cell lines tested. Molecular docking studies suggest that ICA may possibly exhibit its anticancer effect by inhibiting EGFR in A549, Bcr-Abl in K562 and GSK-3β kinase in SW480 cell line. Moreover, ICA showed strong binding avidity for Bcl-2 protein in silico, which could result in induction of apoptosis. Cell cycle analysis revealed that both curcumin and ICA induced concomitant cell cycle arrest at G0/G1 and G2/M phase. Western blot shows that ICA could effectively down regulate the expression of cell cycle protein cyclin D1, while promoting the activation of Caspase 3, 8 and 9 when compared to curcumin in human colon cancer cell line SW480. ConclusionThe result of this study indicates that ICA could hold promise to be a potential anti-cancer agent. Since ICA has shown encouraging results in terms of its anti-cancer activity compared to curcumin, further research is necessary to fully delineate the underlying molecular mechanism of its anticancer potential.

Identification and Characterization of CD133(pos) Subpopulation Cells From a Human Laryngeal Cancer Cell Line.

BackgroundRecent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention.Material/MethodsIn this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line.ResultsUpon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells.ConclusionsAbove all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells.

Eliciting cytotoxic T lymphocytes against human laryngeal cancer-derived antigens: evaluation of dendritic cells pulsed with a heat-treated tumor lysate and other antigen-loading strategies for dendritic-cell-based vaccination.

BackgroundDendritic cells (DCs) have been used successfully in clinical pilot studies. However, tumor-specific immunity and clinical responses were only induced in certain cancer patients. It has been well documented that immunotherapy efficacy can be optimized for responses using antigen pulsing.MethodsThe human laryngeal squamous cell cancer (LSCC) cell line SNU899 was used to evaluate the in vitro anti-tumor efficacy of three different preparations of dendritic cell (DC) vaccines consisting of either whole tumor cells or their derivatives including: i) DCs pulsed with a tumor cell supernatant (DC-TCS), ii) DCs pulsed with whole-cell tumor stressed lysate (DC-TSL), and iii) DCs pulsed with irradiated tumor cells (DC-ITC).ResultsOur results showed that DC-TSL is an effective source of tumor-associated antigens (TAAs) for pulsing DCs. DC-TSL induced the highest expansion of TAA-specific T cells, the strongest Th1 cytokine response, and the most potent cytotoxic T lymphocyte (CTL) activity. DC-TCS and DC-ITC inhibited T cell activation but induced a certain extent of CTL activity.ConclusionsThese data suggest that DC-TSL is a more potent inducer of antitumor immunity against laryngeal cancer than other antigen-loading strategies using whole tumor cell materials. This strategy provides an alternative approach for DC-based immunotherapy for laryngeal cancer.

A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Human esophageal cancer-related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep-2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1-ECRG4 was constructed and introduced into Hep-2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high-level expression of ECRG4. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 over-expression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpres-sion of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl-2-associated X protein, cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase were upregulated in the apoptotic process, whereas B-cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.

Effect of EphA7 Silencing on Proliferation, Invasion and Apoptosis in Human Laryngeal Cancer Cell Lines Hep-2 and AMC-HN-8

Aims: This study aimed to investigate the expression of EphA7 in human laryngeal squamous cell carcinoma (LSCC) tissues and disclose the potential roles and molecular mechanisms of EphA7 in LSCC. Methods: In the present study, we examined EphA7 expression and its function and mechanism in LSCC. EphA7 expression levels were investigated by quantitative real-time PCR (qRT-PCR), western blotting, and immunohistochemistry in a panel of 35 LSCC patient cases. To investigate the potential mechanism of EphA7 in human laryngeal cancer, we employed EphA7 siRNA to knockdown EphA7 expression in LSCC cell line Hep-2 and AMC-HN-8. Subsequently, MTT, TUNEL, qRT-PCR, and western blotting were performed to disclose the roles of EphA7 on proliferation, invasion and migration, and apoptosis in LSCC cell line Hep-2 and AMC-HN-8. Results: Depletion of EphA7 remarkably inhibited the proliferation and invasion of Hep-2 and AMC-HN-8 cells in comparison to control and EphA7 siRNA negative control (NC)-transfected cells. TUNEL staining assay demonstrated that, compared with the control group, the rate of apoptosis in the EphA7 siRNA group was significantly increased. In addition, knockdown of EphA7 in Hep-2 or AMC-HN-8 cells markedly decreased the expression of EphA7 and PTEN, which could contribute to apoptosis. However, the bpV(phen), a PTEN inhibitor, could attenuate anti-proliferation and pro-apoptotic effects of EphA7 siRNA in Hep-2 and AMC-HN-8 cells. Conclusion: Up-regulation of EphA7 was observed in human LSCC samples and down-regulation of EphA7 effectively suppressed laryngeal carcinoma cell growth and promoted its apoptosis. Thus, EphA7 has a critical role in modulating cell growth and apoptosis, which serves as a potential therapeutic target in human LSCC.

Overexpression of SOX2 promotes migration, invasion, and epithelial-mesenchymal transition through the Wnt/β-catenin pathway in laryngeal cancer Hep-2 cells.

SOX2 is a high-mobility group box containing transcription factor essential for the maintenance of embryonic stem cells. Recent evidence indicates that SOX2 overexpression correlates with metastasis and poor prognosis in patients with laryngeal squamous cell cancer. To investigate how SOX2 contributes to this aggressive phenotype, we introduced the human SOX2 gene into a low SOX2-expressing human laryngeal cancer cell line Hep-2. Cell migration and invasion were determined by the Transwell assay with or without Matrigel coating. The epithelial-mesenchymal transition (EMT)-related markers were assayed by Western blot analysis or immunofluorescence. Our results showed that exogenous expression of SOX2 in Hep-2 cells substantially promoted their migratory and invasive capabilities in culture. Moreover, Hep-2 cells stably overexpressing SOX2 underwent EMT phenotype, as evidenced by mesenchymal morphology, decreased expression of epithelial marker (E-cadherin), and increased expression of mesenchymal markers (N-cadherin, vimentin, fibronectin, and α-smooth muscle actin). Strikingly, Western blot analysis and immunofluorescence also showed that overexpression of SOX2 resulted in substantial increase and nuclear accumulation of β-catenin in Hep-2 cells. However, small interfering RNA targeting β-catenin significantly attenuated the reduced expression of E-cadherin and increased cell migration and invasion abilities in SOX2-overexpressing cells, suggesting that SOX2-induced EMT process, migration, and invasion are dependent on β-catenin activation. Taken together, our findings underscore a novel role for SOX2 in laryngeal cancer migration and invasion.

Chloroquine synergizes sunitinib cytotoxicity via modulating autophagic, apoptotic and angiogenic machineries

Tyrosine kinases play a pivotal role in oncogenesis. Although tyrosine kinase inhibitors as sunitinib malate are used in cancer therapy, emerging studies report compromised cytotoxicity when used as monotherapy and thus combinations with other anti-cancer agents is recommended. Chloroquine is a clinically available anti-malarial agent which has been shown to exhibit anti-cancer activity. In the current study, we questioned whether chloroquine can modulate sunitinib cytotoxicity. We found that chloroquine synergistically augmented sunitinib cytotoxicity on human breast (MCF-7 and T-47D), cervical (Hela), colorectal (Caco-2 and HCT116), hepatocellular (HepG2), laryngeal (HEp-2) and prostate (PC3) cancer cell lines as indicated by combination and concentration reduction indices. These results were also consistent with that of Ehrlich ascites carcinoma (EAC) Swiss albino mice models as confirmed by tumor volume, weight, histopathological examination and PCNA expression. Sunitinib induced autophagy via upregulating beclin-1 expression which was blocked by chloroquine as evidenced by accumulated SQTSM1/p62 level. Furthermore, chloroquine augmented sunitinib-induced apoptosis by decreasing survivin level and increasing caspase 3 activity. Chloroquine also enhanced the antiangiogenic capacity of sunitinib as indicated by decreased CD34 expression and peritoneal/skin angiogenesis. Sunitinib when combined with chloroquine also increased reactive nitrogen species production via increasing inducible nitric oxide synthase expression and nitric oxide level whilst reduced reactive oxygen species production by increasing GSH level, activities of glutathione peroxidase and catalase and reducing lipid peroxides compared to sunitinib-only treated group. Taken together, these findings suggest that chloroquine enhanced sunitinib cytotoxicity in a synergistic manner via inducing apoptosis while switching off autophagic and angiogenic machineries. Nevertheless, further studies are required to elucidate the efficacy and safety profile of such combination.

EGFR-targeted poly(ethylene glycol)-distearoylphosphatidylethanolamine micelle loaded with paclitaxel for laryngeal cancer: preparation, characterization and in vitro evaluation

The objective of this study was to evaluate the potential of using polymeric micelles modified with a peptide (termed GE11) ligand of epidermal growth factor receptor as the targeted carriers to achieve increased accumulation in laryngeal cancer and enhanced intracellular delivery for the encapsulated anticancer drugs. Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) micelles containing paclitaxel were prepared via film-hydration method followed by investigation of in vitro release of paclitaxel in phosphate-buffered saline. The average size of GE11-PEG-DSPE/paclitaxel micelle and mPEG-DSPE/paclitaxel were 35 ± 2.8 nm [the polydispersity index (PDI) = 0.207] and 28 ± 2.1 nm (PDI = 0.154), respectively. Micelles with or without GE11-modified had similar physicochemical properties. Transmission electron microscopy showed that the micelles were homogeneous and spherical in shape. Encapsulation efficiency and drug loading of the micelle were 74.11 ± 3.89% and 3.58 ± 2.82%, respectively. The in vitro targeting characteristic of GE11-modified micelles was investigated by observing the level of cellular uptake of fluorescent coumarin-6-loaded micelles on EGFR over-expressed human laryngeal cancer cell line Hep-2 and EGFR low-expressed human leukemic cell line U-937. Hep-2 cell proliferation was significantly inhibited by GE11-PEG-DSPE/paclitaxel micelle compared to mPEG-DSPE/paclitaxel micelle and Taxol in vitro. Our results suggested that GE11-PEG-DSPE micelle could be a promising strategy for enhancing paclitaxel’s chemotherapeutic effects on EGFR over-expressed cancer cells.

Low-energy ion beam bombardment of human cancer cells in vacuum to induce DNA transfection

A process of low-energy ion-beam-induced gene transfection in human cancer cells was developed for non-viral DNA transfection. The whole process included maintenance of the cells in vacuum, ion beam bombardment of the cells with optimized ion beam condition leading to a medium cell viability, and DNA transfer. A method of using a specially designed liquid-nitrogen cooled sample holder to house the cells and frozen medium to cover and protect the cells was adopted to maintain the cells viable in the vacuum condition during ion beam bombardment. Nitrogen ion beams with energy in the range 7–28 keV were used to bombard human cancer cells (HEp-2, the human laryngeal epitheloid cancer cell line) to fluence orders of 1015–1016 ions/cm2. After ion beam bombardment, the cell viability was studied. The ion beam condition which resulted in a relatively medium viability of the cells was chosen to operate ion bombardment to induce gene transfection. To the viable cells, DNA transfection using plasmid DNA pEGFPN2 was carried out. The experiment demonstrated that under appropriately controlled ion beam conditions and freezing environment, the human cells could survive and be transfected with exogenous DNA. This technique could be used as an alternative of non-viral gene transfections with a promising efficiency.

Knockdown of ZNF403 inhibits cell proliferation and induces G2/M arrest by modulating cell-cycle mediators

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.

Suppression of Aurora-A by RNA interference inhibits laryngeal cancer Hep-2 cell growth

To investigate the effects of knockdown of Aurora-A by RNA interference on laryngeal cancer Hep-2 cell growth in vitro and in vivo. A plasmid containing siRNA against Aurora-A was constructed and transfected into human laryngeal cancer cell line Hep-2. Measurements included the CCK-8 assay for viability and proliferation, Transwell assay for invasion, colony formation assay for cell anchorage-independent growth. Western blot and immunohistochemistry assay for protein expression. Tumorigenicity was observed in vivo. In Hep-2 cells transfected by Aurora-A siRNA (designated as siRNA-3), protein expression of Aurora-A was suppressed by 52%. In CCK-8 assay, absorbance value of siRNA-3 cells (3.268 ± 0.106, (x(-) ± s)) was lower than that of Hep-2 cells (3.722 ± 0.152, F = 17.634, P < 0.001). In Transwell assay, the average invasive cells per field in siRNA-3 cells (110.0 ± 18.0) was less than that in Hep-2 cells (236.0 ± 26.0, F = 26.462, P < 0.01). In colony formation assay, the average colony number of siRNA-3 cells (31.0 ± 6.6) was lower than that of Hep-2 cells (104.0 ± 14.0). The average tumor size in siRNA-3 group was (127.77 ± 174.83) mm(3), which was less than Hep-2 cell group (837.26 ± 101.80) mm(3), (F = 28.187, P < 0.001). Silencing of Aurora-A decreased the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion. The knockdown of Aurora-A inhibits the growth and invasiveness of Hep-2 cells in vitro and in vivo, which may be a promising therapeutic strategy for LSCC.

Antiplasmodial activity of botanical extracts against Plasmodium falciparum

The absence of a vaccine and the rampant resistance to almost all antimalarial drugs have accentuated the urgent need for new antimalarial drugs and drug targets for both prophylaxis and chemotherapy. The aim of the study was to discover effective plant extracts against Plasmodium falciparum. In the present study, the hexane, chloroform, ethyl acetate, acetone, and methanol extracts of Citrus sinensis (peel), Leucas aspera, Ocimum sanctum, Phyllanthus acidus (leaf), Terminalia chebula (seed) were tested for their antimalarial activity against chloroquine (CQ)-sensitive (3D7) strain of P. falciparum which was cultured following the candle-jar method. Antimalarial evaluations of daily replacement of culture medium containing CQ and different plant crude extracts were performed on 96-well plates at 37°C for 24 and 48 h. Parasitemia was determined microscopically on thin-film Giemsa-stained preparations. Plant extracts were tested for their cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on human laryngeal cancer cell line (HEp-2) and normal cell line (Vero). Out of the 25 extracts tested, six showed good (IC(50) 4.76-22.76 μg/mL), 15 exhibited moderate (IC(50) 31.42-88.03 μg/mL), while four displayed mild (IC(50) > 100 μg/mL) antiplasmodial activity. The leaf ethyl acetate and methanol extracts of L. aspera; ethyl acetate, acetone, and methanol extracts of P. acidus; and seed acetone extract of T. chebula had good antiplasmodial activity (IC(50) = 7.81, 22.76, 9.37, 14.65, 12.68, and 4.76 μg/mL) with selectivity indices 5.43, 2.04, 4.88, 3.35, 3.42, and 9.97 for HEp-2 and >5.79, >2.20, >11.75, >3.41, >3.94, and >7.38 for Vero cells, respectively. These analyses have revealed for the first time that the components present in the solvent extracts of L. aspera, P. acidus, and T. chebula have antiplasmodial activity. The high antiplasmodial activity observed make these plants good candidates for isolation of anti-protozoal compounds which could serve as new lead structures for drug development.

The impact of 60Co γ-ray on apoptosis to Hep-2 human laryngeal cancer cell in the condition of reoxygenation after hypoxia

Objective The aim of the study was to establish models of Hep-2 laryngeal cancer cell line of different oxygen supplying, trying to investigate the impact of normoxia, hypoxia, reoxygenation after hypoxia on apoptosis and expression of proteins HIF-1α and p53 to Hep-2 human laryngeal cancer cell line induced by 60Co γ-ray.

Increased expression of telomere-related proteins correlates with resistance to radiation in human laryngeal cancer cell lines

Telomere-associated proteins function as survival factors in telomere maintenance, which are major contributors to radiosensitivity in human cancers. The aim of this study was to investigate the association of telomere-associated gene expression and radiation resistance in human larynx squamous carcinoma. The changes of telomere-associated gene expressions and bionomical characteristics that occur in two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were explored in the present study. Based on previous research, elevated POT1 and TPP1 expressions were detected by reverse transcription-PCR and Western blotting in Hep-2R cell lines. Furthermore, Hep-2R cells showed increased recovery ratio accompanied by a reduction of cell arrested in G2/S phase, suggesting that the radioresistance of Hep-2R cells was due to a faster growth in which telomere length had recently been demonstrated to be a powerful prognostic marker. These results manifest that radioresistant Hep-2R cell lines showed certain changes in gene expression and bionomical profiles that are different from the profile changes of the more-sensitive Hep-2 cell lines, and that evaluation of telomere-associated genes may be a prognostic marker for response to radiotherapy in larynx squamous cell carcinoma (LSCC).

Inhibition of cell proliferation and arrest of cell cycle progression by blocking chloride channels in human laryngeal cancer cell line Hep-2

Chloride channel (ClC) is involved in normal physiological processes and pathology of various diseases. Although it is recognized that blockade of ClC inhibits the cell proliferation, it is not well understood the potential function of ClC in laryngeal cancer. In this study, we investigated the effect of the ClC inhibitor on cell proliferation, cell cycle progression in human laryngeal cancer cell line Hep-2, as well as the effect on the phosphorylation levels of ERK1/2 and AKT1. In this study crystal violet method was used to study the effect of the ClC inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid, NPPB, on Hep-2 cell proliferation. The impaction of the inhibitor on the cell cycle distribution was investigated by the flow cytometry (FCM). Western blot was performed to measure the phosphorylation levels of ERK1/2 and AKT1. Our data indicated ClC played an important role in Hep-2 cell proliferation and cell cycle. NPPB inhibited Hep-2 cell proliferation when compared with the controls. Blockade of ClC arrested cell cycle progression and suppressed the phosphorylation of ERK1/2 and AKT1 in Hep-2 cells by inhibition of cell proliferation by CIC inhibitor (NPPB) could be through arresting cell cycle progression, which is probably by suppressing phosphorylation of ERK1/2 and AKT1.

Combined antitumor activity of cucurbitacin B and docetaxel in laryngeal cancer

Combination therapy with multiple drugs is a common practice in the treatment of cancer. The promising clinical activity of docetaxel has promoted considerable interest in combining it with other antitumor agents. To determine whether cucurbitacin B can enhance chemosensitivity to docetaxel in laryngeal cancer, in the present study, we investigated the combined antitumor effect of cucurbitacin B with docetaxel on Hep-2, a human laryngeal cancer cell line. We treated Hep-2 cells with cucurbitacin B alone or in combination with docetaxel and evaluated cell growth, cell cycle distribution, and apoptosis using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay, flow cytometry, and fluorescent microscopy. Our results showed that, in comparison with single agent treatment, the combination of cucurbitacin B and docetaxel produced greater efficacy in growth inhibition, cell cycle arrest at G 2/M phase, and apoptosis induction. Measuring the modulation of regulators in the cell cycle, apoptosis and signal transductions by Western blot analysis showed that the combination effect of cucurbitacin B and docetaxel was due to suppress the expression of p-STAT3 (signal transducers and activators of transcription 3), Bcl-2, and cyclin B1. Moreover, our in vivo studies were reproduced in a mouse xenograft model, where, the combination of cucurbitacin B with docetaxel synergestively inhibited tumor growth. Together, this investigation suggests that cucurbitacin B combined with docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with laryngeal cancer.