Laryngeal Carcinoma Cell Line Hep-2 Research Articles

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.

Genetic Interpretation of the Impacts of Honokiol and EGCG on Apoptotic and Self-Renewal Pathways in HEp-2 Human Laryngeal CD44high Cancer Stem Cells

Most current larynx cancer therapies are generally aimed at the global mass of tumor, targeting the non-tumorigenic cells, and unfortunately sparing the tumorigenic cancer stem cells (CSCs) that are responsible for sustained growth, metastasis, and chemo- and radioresistance. Phytochemicals and herbs have recently been introduced as therapeutic sources for eliminating CSCs. Therefore, we assessed the anti-tumor effects of two herbal ingredients, the green tea extract “Epigallocatechin-3-gallate (EGCG)” and Honokiol (HNK), on parental cells or CD44high CSCs of the human laryngeal squamous cell carcinoma cell line HEp-2. Results revealed that EGCG had a preeminent apoptotic potential on HEp-2 laryngeal CSCs. HNK conferred higher cytotoxic impacts on parental cells mostly by necrosis induction, especially with higher doses, but apoptosis induction with lower doses was also observed. The Notch signaling pathway genes were more potently suppressed by EGCG than HNK. However, HNK surpassed EGCG in downregulating the β-catenin and the Sonic Hedgehog signaling pathways genes. On a genetic basis, both agents engaged the BCL-2 family-regulated and caspase-dependent intrinsic apoptotic pathway, but EGCG and HNK triggered apoptosis via p53-independent and p53-dependent pathways, respectively. Taken together, EGCG and HNK eradicated HEp-2 human larynx cancer cells through targeting multiple self-renewal pathways and activating diverse cell death modalities.

Exploring the Mechanism of MicroRNA-125a Reversing the Resistance of Laryngeal Cancer Stem Cells to Cisplatin from Mitochondrial Autophagy

To explore the mechanism of microRNA-125a reversing the resistance of laryngeal cancer stem cells to cisplatin from mitochondrial autophagy. The human laryngeal carcinoma cell line Hep-2 was purchased from Shanghai Bioleaf Biotechnology Co., Ltd. The expression of microRNA-125a messenger RNA in each group of cells was analyzed by reverse transcription-polymerase chain reaction. The cell migration and invasion ability of each group was evaluated by Transwell assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was conducted to evaluate the protein expression. The expression of microRNA-125a messenger RNA in the Hep-2/diamminedichloroplatinum group was lower than that in the control group (p<0.05) and the expression of microRNA-125a messenger RNA in the microRNA-125a mimic group was higher than that in the Hep-2/diamminedichloroplatinum group (p<0.05). Compared with the Hep-2/diamminedichloroplatinum group, the microRNA-125a mimic group had more mitochondrial fragments and the average branch length of mitochondria decreased (p<0.05). The cell migration, invasion and viability of the Hep-2/diamminedichloroplatinum group increased (p<0.05) and the apoptosis rate decreased (p<0.05). The protein expression of phosphoinositide 3-kinase, protein kinase B and mammalian target of rapamycin in the Hep-2/diamminedichloroplatinum group was higher than that in the control group (p<0.05) and their expression in the microRNA-125a mimic group was lower than that in the Hep-2/ diamminedichloroplatinum group (p<0.05). The dysregulation of microRNA-125a was related to diamminedichloroplatinum resistance in laryngeal cancer and diamminedichloroplatinum resistance was induced by microRNA-125a through phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway mediated autophagy inhibition. Overexpression of microRNA-125a inhibited the growth and metastasis of Hep-2/diamminedichloroplatinum cells through the phosphoinositide 3-kinase/ protein kinase B/mammalian target of rapamycin pathway and induced their apoptosis and autophagy.

Evaluation of the Effect of Matrine on Human Laryngeal Squamous Cell Carcinoma Cell Line

Aim of study: to evaluate the possible anticancer potential of Matrine, one of the main active components extracted from dry roots of Sophora flavescens Ait (Leguminosae), on human laryngeal squamous cell carcinoma cell line (Hep-2). Material and methods: Prepared (Hep-2) cell line was treated with different concentrations of Matrine for 48 hours. The effect of Matrine on cell line was investigated using MTT assay, cytological examination and nuclear morphometric analysis of treated cells to explore the effect of the drug on cell proliferation and induction of apoptosis. The results were analyzed statistically. P value ≤ 0.05 was considered significant. Results: MTT assay showed that the proliferation of Matrine treated cells (in relation to control cells) decreased in a dose dependent manner. Morphometric analysis showed a decrease in nuclear area factor (NAF), which is an indicator of apoptosis, as concentrations increased. The results were statistically significant. Conclusion: Matrine has a cytotoxic effect on Hep-2 cell line through induction of apoptosis.

Dihydroartemisinin Prevents Distant Metastasis of Laryngeal Carcinoma by Inactivating STAT3 in Cancer Stem Cells.

BackgroundAccumulating evidence indicates that cancer stem cells (CSCs) are a minor subpopulation of cancer cells that may be the primary source of cancer invasion, migration, and widespread metastasis.Material/MethodsWe investigated the effects of dihydroartemisinin (DHA) on distant metastasis of laryngeal carcinoma and the relevant mechanism. In vitro, we used the Hep-2 human laryngeal squamous carcinoma cell line (Hep-2 cells) to assemble CSCs, using CD133 as the cell surface marker. Our data demonstrate that the CD133+ subpopulation of Hep-2 cells has greater invasion and migration capabilities than CD133− cells. We also evaluated the effects of DHA, a newly defined STAT3 inhibitor, on the invasion and migration of CD133+ Hep-2 cells under hypoxia and IL-6 stimulation, both of which can activate STAT3 phosphorylation.ResultsCSCs exhibited a significant decrease in the ability of migration and invasion upon the application of DHA, along with simultaneous alterations in related proteins, both in cultured cells and in xenograft tumors. The associated signaling proteins included phosphorylated STAT3 (p-STAT3), matrix metalloproteinase-9 (MMP-9), and E-cadherin, which are closely involved in cancer invasion and metastasis. In vivo, we found that DHA can reduce lung metastasis formation caused by CSCs and prolong survival in mice, and can inhibit STAT3 activation, downregulate MMP-9, and upregulate E-cadherin in lung metastatic tumors.ConclusionsTaken together, our findings indicate that CSCs possess stronger invasive and metastatic capabilities than non-CSCs, and DHA inhibits invasion and prevents metastasis induced by CSCs by inhibiting STAT3 activation.

AMG-102 inhibits proliferation and induces apoptosis of laryngeal squamous cell carcinoma cells by regulating c-Met/PI3K/Akt pathway

Objective: To investigate the effects of c-Met inhibitor AMG-102 on the proliferation and apoptosis of laryngeal squamous carcinoma Hep-2 cells and the underlying mechanism. Methods: Laryngeal squamous carcinoma cell line Hep-2 cells were treated with 2.5, 5 and 10 μmol/L AMG-102, respectively. The proliferation activities of Hep-2 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). The apoptotic rate of Hep-2 cells was detected by flow cytometry analysis and Hoechst staining. The mRNA expression levels of apoptosis-related genes were detected by real-time quantitative polymerase Chain reaction (RT-qPCR), and the protein expressions of c-Met/PI3K/AKT pathway were detected by western blot. Results: Compared with the control group, the proliferation rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 24 hours were (89.8±1.1)%, (79.8±1.0)% and (69.1±1.2)%, respectively; for 48 hours were (76.8±2.0)%, (60.2±1.1)% and (49.8±1.2)%, respectively; for 72 hours were (50.1±2.0)%, (41.5±1.1)% and (33.6±1.0), respectively, with significant differences (all P<0.05). The apoptotic rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours were (16.09±1.53)%, (27.51±2.02)% and (36.57±1.42)%, respectively, which were significantly higher than (3.62±0.10) % in the control group (all P<0.05). After treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours, the relative expression levels of Bcl-2 mRNA in Hep-2 cells were 0.58±0.13, 0.38±0.12 and 0.20±0.13, respectively; the relative protein expression of p-Met were 80.0±3.8, 50.6±4.2 and 28.5±1.3, respectively; the relative protein expression of p-PI3K were 87.1±0.9, 54.2±1.2 and 21.0±1.2, respectively; the relative protein expression of p-AKT were 98.7±5.6, 56.9±3.2 and 32.2±4.3, respectively; which were significantly lower than those in the control group (all P<0.05). The relative expression levels of Bax mRNA were 1.78±0.13, 2.37±0.14 and 3.05±0.13, respectively, and the relative expression levels of caspase-3 mRNA were 1.98±0.14, 2.47±0.14 and 3.15±0.13, respectively, which were significantly higher than those in the control group (all P<0.05). Conclusion: c-Met inhibitor AMG-102 could inhibit the proliferation and induce apoptosis of laryngeal squamous carcinoma Hep-2 cells by regulating the c-Met/PI3K/Akt pathway.

Effect of c-Met inhibitor AMG-102 on radiosensitivity in laryngeal squamous carcinoma cells

Objective: To investigate the effect of c-Met inhibitor AMG-102 on proliferation and radiosensitivity in laryngeal squamous carcinoma cells. Methods: The effects of AMG-102 on proliferation and radiosensitivity of laryngeal squamous carcinoma cell lines Hep-2 and KBV200 were detected by 3-(4, 5-dimethy-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and colony formation assay, respectively. The apoptosis of Hep-2 and KBV200 cells was detected by flow cytometry. The expression levels of c-Met, phospho-Met (p-Met), cleaved caspase-3 and Akt/p-Akt, Erk/p-Erk were detected by Western blot. Specific small interfering RNA targeting c-Met or plasmid of c-Met were transfected into Hep-2 and KBV200 cells to investigate the cell sensitivity to AMG-102. Results: Compared with KBV200 cells, Hep-2 cells were more sensitive to AMG-102 with IC(50) of 14 and 9 μmol/L, respectively. The relative expression levels of c-Met and p-Met proteins in Hep-2 cells were 194.48±0.57 and 177.76±1.53, respectively, which were significantly higher than those in KBV200 cells (171.24±1.00 and 115.37±0.56, respectively, P<0.001 for both). Exogenous hepatocyte growth factor (HGF) was added to increase the expression level of p-Met protein in KBV200 cells. The results showed that AMG-102 significantly reduced the expression of p-Met in KBV200 cells treated with HGF (P<0.001). Compared with the dimethyl sulfoxide (DMSO) group, AMG-102 treatment combined with radiotherapy significantly increased the radiosensitivity of Hep-2 cells (SER=1.28, P<0.001). However, AMG-102 had little effect on the radiosensitivity of KBV200 cells (SER=1.18, P=0.002). Compared with the 4 Gy radiotherapy alone group and the 5 μmol/L of AMG-102 alone treatment group, the apoptosis rate of Hep-2 cells in the combined treatment group was significantly increased. Meanwhile, the expression level of cleaved caspase-3 protein was also markedly increased. However, there were no significant changes in the apoptotic rate and cleaved caspase-3 expression in each treatment group of KBV200 cells. Compared with DMSO treatment group, the expression levels of p-Met, p-Akt and p-Erk were significantly decreased in the 4 Gy radiotherapy group, 5 μmol/L of AMG-102 treatment group and combined treatment group of Hep-2 cells. And the levels of p-Met, p-Akt and p-Erk in the combined treatment group were significantly lower than those in the 4 Gy radiotherapy alone group and 5 μmol/L of AMG-102 treatment alone group. By contrast, in KBV200 cells, the expression of p-Met, p-Akt and p-Erk in each group was not changed. The relative expression of p-Met in Hep-2 cells before and after radiotherapy at 30 min, 1 h, 4 h, 8 h, 24 h were 99.89±0.61, 138.62±1.00, 163.07±5.00, 87.80±1.85, 90.67±0.65 and 94.09±1.41, respectively. The level of p-Met was slightly increased after radiotherapy at 30 min and 1 h (P<0.001 for all), whereas it was significantly decreased from 4 h to 24 h after radiotherapy (P<0.05 for all). By contrast, the expression of p-Met in KBV200 cells did not change with time after radiotherapy (P>0.05). The sensitivity of Hep-2 cells to AMG-102 was decreased after silencing of c-Met, while the sensitivity of KBV200 cells to AMG-102 was not significantly changed (P>0.05). Moreover, the radiosensitivity of Hep-2 cells in c-Met knockdown group had a slightly increasing trend (SER=1.07, P=0.068). After the treatment with 10 μmol/L of AMG-102, the proliferation rate of c-Met ectopically expressed KBV200 cells was 60.05%±3.23%, It was significantly lower than that of the blank control 90.08%±1.04% and siRNA negative control (90.12%±1.01%, P<0.001). The results suggested that the overexpression of c-Met in KBV200 cells increased the radiosensitivity to AMG-102, whereas depletion of c-Met resulted in resistance to AMG-102 in Hep-2 cells. Furthermore, the radiosensitivity of KBV200 cells that overexpressed c-Met showed a decreased trend (SER=0.7, P=0.005). Conclusions: c-Met inhibitor AMG-102 has a significant inhibitory effect on the proliferation of c-Met overexpressing laryngeal squamous carcinoma cells, leading to increased radiosensitivity. It suggests that molecular targeted therapy against c-Met receptor is more effective in c-Met overexpressed subtype of laryngeal squamous cell carcinoma.

Role of SATB1 in invasion and metastasis of human laryngeal squamous cell carcinoma cell line Hep-2 and its mechanisms

Objective To investigate the role of special AT-rich sequence-binding protein 1 (SATB1) in invasion and metastasis of human laryngeal squamous cell carcinoma cell line Hep-2 and its mechanism. Methods Hep-2 cells were cultured in vitro. The cell lines were divided into two groups. The cells in the experimental group was transfected with Lipofectamine 2000 liposome for SATB1 small interfering RNA (SATB1-siRNA), and the cells in the control group was transfected with Lipofectamine 2000 liposome for NC-siRNA. The before and after transfection SATB1, E-cadherin and Snail genes relative expression levels between the two groups were detected and compared using RT-PCR. Transwell cell invasion experiments were applied to evaluate the cells invasive ability of the two groups. The scratch experiments were applied to evaluate the 48 h cell migration ability of the two groups. Results The differences in the relative expression level of SATB1, E-cadherin and Snail genes in pre-transfected laryngeal squamous cell line Hep-2 between the two groups were not statistically significant (all P>0.05). Compared with the control group, the relative expression levels of the mRNA of SATB1 and Snail genes decreased in the experimental group after transfection, while the relative expression levels of the mRNA of E-cadherin gene increased, and the differences were statistically significant (all P<0.05). After the transfection, the number of transwell membrane cells and cell migration distances of the two groups of cells decreased. The values of cell number and migration distance in the experimental group were lower than those in the control group, and the differences were statistically significant (all P<0.05). Conclusions SATB1 can promote the invasion and metastasis of Hep-2 cells, and its mechanism may be related to the down-regulation of E-cadherin and the up-regulation of Snail gene expression to induce epithelial-mesenchymal transition. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; Neoplasm metastasis; Special AT-rich sequence-binding protein 1

Soyasapogenol B Attenuates Laryngeal Carcinoma Progression through Inducing Apoptotic and Autophagic Cell Death.

Soyasapogenol B (Soy B), a constituent of soybean, has been shown to exhibit antitumor activities against different types of cancers. However, to our knowledge, no studies so far have investigated the effect of Soy B in human laryngeal carcinoma. This study, therefore, aimed to determine the effect of Soy B in human laryngeal carcinoma cell lines HeP-2 and TU212 and to elucidate the possible underlying mechanisms by which Soy B can induce its antitumor effects. The results showed that Soy B effectively attenuated the cell growth by causing G0/G1 phase cell cycle arrest in laryngeal carcinoma cell lines. Moreover, the percentage of apoptotic and autophagic cells dramatically increased upon exposure to Soy B. Western blotting results confirmed that Soy B can alter the expression levels of established markers of apoptosis and autophagy. Interestingly, both apoptosis inhibitor (ZVAD-fmk) and autophagy inhibitor (3-MA) could partially reverse the effect of Soy B, while blocking autophagy did not cause obvious alteration in the percentage of apoptotic cells. Similarly, in vivo studies validated that Soy B could effectively reduce the size of the tumor and induce apoptosis and autophagy in tumor tissues. Collectively, these results suggested that Soy B can exert anticancer activities against laryngeal carcinoma through inducing apoptotic and autophagic cell death. Our study highlighted the potential role of Soy B as a chemotherapeutic agent for laryngeal carcinoma. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1851-1858, 2020. © 2019 American Association for Anatomy.

Diagnostic value of serum miRNA let-7a for laryngeal carcinoma and effects of let-7a on proliferation and apoptosis of laryngeal carcinoma cells

Objective: To investigate the diagnostic value of serum miRNAlet-7a in laryngeal carcinoma and the effect of let-7a on proliferation and apoptosis of laryngeal carcinoma cells. Methods: Real-time quantitative PCR was used to determine the expression level of serum miRNAlet-7a. The miRNA let-7a mimetic was synthesized and transiently transfected into the laryngeal carcinoma Hep-2 cell line by cationic liposome method. The effects of up-regulation of let-7a expression on laryngeal cancer Hep-2 cells were detected by FCM and MTT assays,respectively. The association of let-7a levels with laryngeal cancer and the diagnostic value for laryngeal cancer were analyzed. Measurement data were taken by t test or analysis of variance; Counting data were analyzed by χ(2) test and Fisher exact probability method. The receiver operating characteristic curve was used to analyze the diagnostic value of let-7a for laryngeal cancer. Results: The relative expression of serum let-7a in healthy subjects was significantly higher than that in patients with laryngeal cancer (0.931±0.094) vs (0.380±0.113) (t=26.507,P<0.01). The relative expressions of serum let-7a in patients with laryngeal cancer before and after surgery were (0.380±0.113) vs(0.493±0.164),with significant difference (t=3.848,P<0.01).The relative expression of serum let-7a was related to lymph node metastasis (t=2.946, P<0.01). There was a positive correlation between the relative expression of let-7a in laryngeal carcinoma and that in serum (r=0.466,P=0.003). After transfection of let-7a mimics, Hep-2 cells showed an increased significant increase in the expression of let-7a (P<0.01), proliferation (P<0.01) and apoptosis (P<0.01). ROC curve analysis showed that the best critical value for relative expression of let-7a in the diagnosis of laryngeal carcinoma was 0.557 with a sensitivity of 0.794,a specificity of 0.727,an area under curve(AUC) of 0.859,and a 95%CI of 0.773-0.926. Conclusions: miRNA let-7a can inhibit the proliferation of laryngeal carcinoma Hep-2 cells and promote apoptosis. Serum let-7a is down-regulated in patients with laryngeal cancer and the level of let-7a is related to lymph node metastasis,which would help early diagnosis and postoperative disease monitoring of laryngeal cancer,but further research is needed.

Effect of NLK on the proliferation and invasion of laryngeal carcinoma cells by regulating CDCP1.

The morbidity and mortality of laryngeal cancer are increasing rapidly, seriously threatening human health. There are several causes of laryngeal cancer, but the exact molecular mechanism is unclear. Finding the molecular targets of laryngeal cancer has become an emerging hot spot. Nemo-like kinase (NLK) is abnormally expressed in tumors, but its role in laryngeal cancer has not been reported. Real Time PCR and Western blot were used to detect NLK mRNA and protein expression in cancer tissues and adjacent tissues of laryngeal cancer patients. The laryngeal carcinoma cell line Hep-2 cells were cultured in vitro and randomly divided into three groups: control group, NC group, and NLK siRNA group followed by the analysis of cell proliferation by MTT assay, Caspase3 activity, and cell invasion by the transwell chamber. MMP-9 and CDCP1 expression was measured by Western blot. NLK mRNA and protein expression was significantly increased in laryngeal carcinoma tissues compared with those in adjacent tissues (p<0.05). NLK siRNA transfection into Hep-2 cells significantly down-regulated NLK expression, inhibited Hep-2 cell proliferation and invasion, increased Caspase-3 activity with statistical differences compared to control group (p<0.05). Down-regulation of NLK expression in Hep-2 cells inhibited MMP-9 expression and decreased CDCP1 expression. NLK is expressed in tumor tissues of patients with laryngeal cancer. The down-regulation of NLK expression may play a role in the proliferation, apoptosis, and invasion of laryngeal carcinoma cells and it is possible by regulating MMP-9 and CDCP1 expression.

Pepsin promotes IL-8 signaling-induced epithelial\u2013mesenchymal transition in laryngeal carcinoma

BackgroundLaryngopharyngeal reflux (LPR), with its increasing morbidity, is attracting considerable attention. In recent years, the causal role between LPR and laryngeal carcinoma has been debated. The main harmful component of LPR is pepsin, which has been shown to induce mucosal inflammation by damaging the mucous membrane. Thus, pepsin is linked to an increased risk of laryngeal carcinoma, although the potential mechanism remains largely unknown.MethodsThe human laryngeal carcinoma cell lines Hep-2 and Tu212 were exposed to different pepsin concentrations and the morphology, proliferation, migration, secretion of inflammatory cytokines, and epithelial–mesenchymal transition (EMT) of the cells were assessed. To evaluate whether interleukin-8 (IL-8) had a causal relationship with pepsin and EMT, an IL-8 inhibitor was used to suppress IL-8 secretion during pepsin exposure and the expression of EMT markers, cell proliferation, and migration were analyzed.ResultsPepsin promoted proliferation, colony formation, migration, and IL-8 secretion of Hep-2 and Tu212 cells in vitro. Furthermore, increased pepsin concentrations changed the morphology of Hep-2 and Tu212 cells; levels of the epithelial marker E-cadherin were reduced and those of mesenchymal markers vimentin and β-catenin and the transcription factors snail and slug were elevated. A similar effect was observed in laryngeal carcinoma tissues using immunohistochemistry. IL-8 level was reduced and EMT was restored when pepsin was inhibited by pepstatin. EMT was weakened after exposure to the IL-8 inhibitor, with significant reduction in pepsin-induced cell proliferation and migration.ConclusionsPepsin may induce EMT in laryngeal carcinoma through the IL-8 signaling pathway, which indicates that it has potential role in enhancing cell proliferation and metastasis of laryngeal carcinoma.

Nuclear Nrf2 Activity in Laryngeal Carcinoma is Regulated by SENP3 After Cisplatin-Induced Reactive Oxygen Species Stress.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a nuclear transcription factor that is activated by reactive oxygen species (ROS). Recent studies reported that hyperactivation of the Nrf2 pathway creates an environment that favors the survival of normal as well as malignant cells, protecting them against oxidative stress, chemotherapeutic agents, and radiotherapy. SUMO1/sentrin/SMT3 specific peptidase 3 (SENP3) reverses sumoylation of small ubiquitin-like modifier (SUMO)-conjugates. We demonstrated that Nrf2 was detected in the nuclei of laryngeal carcinoma cells, but not in cells of tissues surrounding the cancer, which correlated with the appearance of SENP3 in the nuclei. Silencing of Nrf2 in laryngeal carcinoma cell line Hep-2 significantly reduced cell viability and enhanced apoptosis rates under cisplatin, 5-fluorouracil (5-FU) and phenethyl isothiocyanate (PEITC) exposure. Cisplatin exposure induced ROS stress in Hep-2 cells in a time-dependent manner and was accompanied by increased Nrf2 and SENP3 protein accumulations, an effect reversed by the addition of the antioxidant N-acetyl-cysteine (NAC). Silencing of SENP3 led to reduced Nrf2 protein levels, whereas overexpression of SENP3 led to concomitant enhanced transcription of the Nrf2 target genes HO-1, NQO1, GCLC and GSTM1. Immunoprecipitation showed that overexpressed Nrf2 and SENP3 could be precipitated together, indicating that they were intracellular bound to each other. Our data identified intranuclear activation of Nrf2 is triggered by cisplatin-induced ROS development through the activity of SENP3. These findings provide novel insights into the Nrf2 reduced cancer cell response to the chemotherapy of laryngeal carcinoma.

MiR-503 enhances the radiosensitivity of laryngeal carcinoma cells via the inhibition of WEE1.

Enhancing the sensitivity of laryngeal cells to radiation is crucial for improving the efficacy of laryngeal carcinoma. MicroRNAs are known to play a major role in regulating cellular radiosensitivity. This study was designed to explore the effect and the molecular basis of miR-503 in the radiosensitivity of laryngeal carcinoma cells. Quantitative real-time polymerase chain reaction analysis showed that miR-503 expression was decreased in human laryngeal carcinoma cell lines Hep-2 and TU212, and the downregulation of miR-503 was also observed after irradiation. Upregulation of miR-503 by pre-miR-503 transfection restrained proliferation, promoted progression of Hep-2 and TU212 cells through the cell cycle after irradiation, and sensitized cells to radiation. Dual-Luciferase Reporter Assay verified a direct interaction between miR-503 and the WEE1 messenger RNA 3'-untranslated region. The overexpression of miR-503 significantly decreased WEE1 expression at the messenger RNA and protein levels, whereas the inhibition of miR-503 upregulated the expression of WEE1. WEE1 knockdown by WEE1 small interfering RNA apparently abrogated the inhibitory effect of anti-miR-503 on radiosensitivity. In conclusion, miR-503 could function as an enhancer of radiation responses in laryngeal carcinoma cells by inhibiting WEE1, which may be a potential novel radiosensitizing strategy for laryngeal carcinoma.

Radiosensitization by CpG ODN7909 in an epidermoid laryngeal carcinoma Hep-2 cell line.

ObjectiveTo evaluate the radiosensitivity effect of CpG oligodeoxyribonucleotide (ODN) 7909 on human epidermoid cancer strain-2 (Hep-2) cells in vitro and discuss the potential for improved radiotherapy treatment in patients with laryngeal squamous cell carcinoma.MethodsToll-like receptor (TLR) 9 expression was assessed in Hep-2 cells using Western blots and reverse transcription polymerase chain reaction. Cell Counting Kit-8 was used to detect Hep-2 cell viability at 24 and 48 h following treatment with different CpG ODN7909 concentrations. Cellular colonization was evaluated using microscopy. Cell cycle distribution and apoptosis rate was determined with flow cytometry. Interleukin (IL)-12 and tumour necrosis factor (TNF)-α concentrations were detected by enzyme-linked immunosorbent assay.ResultsHep-2 cells were found to express TLR9, and CpG ODN7909 treatment suppressed Hep-2 cell viability in a dose- and time-dependent manner. Cell survival curve analyses revealed a sensitivity enhancement ratio of the mean death dose of 1.225 for CpG ODN7909 plus irradiation versus irradiation alone. Furthermore, the population of Gap 2/mitotic-phase cells, apoptosis rate and secreted IL-12 and TNF-α levels were significantly increased in Hep-2 cells treated with CpG ODN7909 plus irradiation versus IR alone.ConclusionCpG ODN7909 enhanced the radiosensitivity of Hep-2 cells in vitro.

The role of autophagy in the curcumin induced proliferation in human laryngeal cancer Hep2 cell

Objective:The aim of this study is to study the effect of 3-methyladenine (3-MA) on the autophagy and apoptosis of human laryngeal cancer Hep 2 cells induced by curcumin. Method:The proliferation of human laryngeal cancer Hep2 cells was observed by MTT assay. The autophagy level was detected by AO acridine orange staining. Annexin VFITC/PI double staining was used to detect the apoptosis of Hep2 cells. The expression of LC3, Beclin1, Bcl-2 and Bax protein were detected by Western blot. Result:MTT assay showed that curcumin inhibited the proliferation of Hep2 cells in a dose/time dependent manner. The apoptosis rate of curcumin combined with 3-MA increased (P<0.05). Acridine orange staining showed that 3-MA combined with curcumin could significantly reduce the autophagy level of laryngeal carcinoma Hep2 cells. The expression of Bcl-2, Bclin-1 and LC3 Ⅱ was decreased, while the expression of Bax protein was increased (P<0.05). Conclusion:Curcumin can induce apoptosis of Hep 2 cells and induce the development of protective autophagy. The inhibitory effect of curcumin on the apoptosis of laryngeal carcinoma Hep2 cell line was significantly enhanced by 3-MA.

Supporting effect of polyethylenimine on hexarhenium hydroxo cluster complex for cellular imaging applications

Abstract The present work introduces an insight into pH- and concentration-dependent binding of branched polyethylenimine (PEI) with hexarhenium hydroxo cluster complex [{Re 6 S 8 }(OH) 6 ] 4− aimed at enhancing the stability, luminescence intensity and cellular uptake of the cluster complex in aqueous solutions at pH 7.4. Luminescence and conductivity measurements revealed pH 7.4 and 1:1 of PEI:cluster molar ratio as specific conditions ensuring multiple electrostatic interactions of protonated amino groups of PEI with the tetra-anionic cluster units. These interactions resulted in a significant supporting effect of PEI on stability and luminescence of the cluster complex, while no effect of the cluster complex on the acid-base and aggregation properties of PEI was detected from dynamic light scattering and 1 H NMR spectroscopy measurements. The cluster luminescence remained without degradation for at least two weeks due to the supporting effect of PEI, which can facilitate the application of the cluster complex in bioimaging. The cellular uptake of [{Re 6 S 8 }(OH) 6 ] 4− and [{Re 6 S 8 }(OH) 6 ] 4− @PEI was investigated. Confocal microscopic studies showed that the pristine cluster complex [{Re 6 S 8 }(OH) 6 ] 4− did not enter cells (the human larynx carcinoma cell line (Hep-2)), whereas the supramolecular assembly [{Re 6 S 8 }(OH) 6 ] 4− @PEI internalized into the cells.

AgNPs and Ag/C225 Exert Anticancerous Effects via Cell Cycle Regulation and Cytotoxicity Enhancement

Currently, cancer has become the leading cause of deaths and is a serious threat to human health. Nanotechnology-based medical applications are developing rapidly, especially in anticancer research, as nanomaterials can be retained in the cell after their entry across the cell membrane, making them potential candidates for application in the diagnosis and treatment of tumours. Here, we prepared silver nanoparticles (AgNPs) and silver/cetuximab (Ag/C225) particles of approximately 20 nm and investigated their inhibitory effect on proliferation of human nasopharyngeal carcinoma cell line CNE and laryngeal carcinoma cell line HEp-2. The MTT assay was used to determine their half-maximal inhibitory concentration (IC50) values. Electron microscopy revealed no obvious morphological differences between the prepared AgNPs and Ag/C225. Treatment of both cell lines with AgNPs and Ag/C225 increased the proportion of cells in the G0/G1 stage and decreased the proportion of cells in the S stage. AgNP and Ag/C225 treatments increased the expression of apoptotic proteins Bax and P21 and decreased the expression of Bcl-2 in CNE cells, compared to control cells. Ag/C225 is internalised by CNE cells, and it enhances X-irradiation-induced cytotoxicity. These results help increase the understanding of the anticancer effect of AgNPs and Ag/C225 and explore their application as radiosensitisers.

Identification and characterization of CD133+CD44+ cancer stem cells from human laryngeal squamous cell carcinoma cell lines.

Background: Laryngeal squamous cell carcinoma ranks second among head and neck squamous-cell carcinomas. Cancer stem cells can support cancer growth and malignant behavior. Therefore, cancer stem cells isolated from laryngeal squamous cell carcinoma tissue could be used to investigate the initiation, progression, and treatment strategies of this cancer. Methods: We isolated CD133-CD44-, CD133-CD44+, CD133+CD44- and CD133+CD44+ cell populations from laryngeal squamous-cell carcinoma cell lines Hep2 and TU-177 by magnetic-activated cell sorting. Sphere formation, cell proliferation, migration, invasion, colony formation, resistance to radio- and chemotherapy, and in vivo tumorigenicity of these populations were evaluated. Moreover, we investigated the expression of the stem-cell markers (sex determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) in CD133-CD44-, CD133-CD44+, CD133+CD44-, CD133+CD44+ cell populations and parental Hep2 and TU-177 cells. Results: As compared with CD133-CD44-, CD133-CD44+, CD133+CD44- populations and parental cells, CD133+CD44+ cells showed higher cell viability, migration and invasive capability and colony formation ability as well as stronger resistance to cisplatin and irradiation. Moreover, levels of SOX2 and OCT4 and tumorigenicity in nude mice were greater in CD133+CD44+ Hep2 and TU-177 cells than other cell populations and parental cells.Conclusion: The CD133+CD44+ population of laryngeal squamous-cell carcinoma Hep2 and TU-177 cells have stem cell properties and showed more malignant features than CD133+CD44- and CD133-CD44+ cell populations. CD133+CD44+ cancer stem cells may be a promising target for developing anticancer drugs and treatment strategies for laryngeal squamous cell carcinoma.

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.