Human Laryngeal Carcinoma Hep-2 Cells Research Articles

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Hexokinase 2 overexpression promotes the proliferation and survival of laryngeal squamous cell carcinoma

Proliferating cancer cells preferentially use anaerobic glycolysis rather than oxidative phosphorylation for energy production. Hexokinase 2 (HK2) is highly expressed in many malignant cells and is necessary for anaerobic glycolysis. The role of HK2 in laryngeal squamous cell carcinoma (LSCC) is unknown. In this study, the expression of HK2 in LSCC was investigated and the effect of inhibiting HK2 expression with small hairpin RNA (shRNA) on tumor growth was investigated. Using immunohistochemistry, HK2 expression was assessed in LSCC tissues. Human laryngeal carcinoma Hep-2 cells were stably transfected with a plasmid expressing HK2 shRNA (pGenesil-1.1-HK2) and were compared to control cells with respect to the cell cycle, cell viability, apoptosis, and their ability to form xenograft tumors. HK2 expression was significantly higher in LSCC than in papilloma or glottis polypus. Tumor samples of higher T, N, and TNM stage often had stronger HK2 staining. HK2 shRNA reduced HK2 mRNA, protein levels, and HK activity in Hep-2 cells. HK2 cells expressing shRNA demonstrated a higher G0-G1 ratio, increased apoptosis, and reduced viability. Xenograft tumors derived from cells expressing HK2 shRNA were smaller and had lower proliferation than those from untransfected or control-plasmid-transfected cells. In conclusion, depletion of HK2 expression resulted in reduced xenograft tumor development likely by reducing proliferation, altering the cell cycle, reducing cell viability and activating apoptosis. These data suggest that HK2 plays an important role in the development of LSCC and represents a potential therapeutic target for LSCC.

The impact on secreted VEGF of Hep-2 human laryngeal cancer cell induced by Rg3

Objective The aim of this study was to investigate the affecting of Rg3 to secreted VEGF of human laryngeal carcinoma Hep-2 cells and its mechanism of inhibition to tumor angiogenesis.

Investigation on biological characteristics of CD133+ cancer stem cells in human laryngeal carcinoma Hep-2 cell line

To investigate the biology characters of CD133+ cancer stem cells from Hep-2 cell line. The Flow cytometry was applied to purify CD133+ cells from Hep-2 cell line. The sorted CD133+ cells were cultured in RPMI1640. The ability of migration, invasion and clonality of CD133+ cells were performed to characterize the properties of the cells. CD133+ cells and CD133- control cells were treated with paclitaxel and exposed respectively to X-rays emitted by linear accelerator with a dose of 10 Gy. The surviving rates and growth inhibition ratio of cells in two groups were detected with MTT assay to observe the resistance to irradiation and chemotherapy in CD133+ cells. The percentage of CD133+ cells in the unsorted and the sorted cells were 3.1% +/- 0.2% and 90.2% +/- 5.5%, respectively. CD133+ cells showed the higher proliferation and colony ability than CD133- cells. The numbers of CD133+ and CD133- cells that passed the membrane of Transwell chamber were 526 +/- 39 and 220 +/- 20 respectively (t = 22.08, P < 0.001). The colony forming units of three passages were 30.0% +/- 4.7%, 32.2% + 3.6%, 32.7% + 3.4% in CD133 cells and 15.2% +/- 2.2%, 12.0% + 2.5%, 13.8% +/- 3.3% in CD133- cells. There were statistic difference between two groups (t = 8.99, t = 14.66, t = 12.69, P < 0.01). At 24, 48, and 72 hours of treatment with paclitaxel, the cell surviving rates of CD133+ cells were 90.1% +/- 5.9%, 85.1% +/- 7.1% and 70.3% +/- 6.4% and lower than those of CD133- cells, respectively (t = 5.24, t = 8.18, t = 8.14, P < 0.01) . After radiotherapy, growth inhibition ratio of CD133+ and CD133- cells were 30.0% +/- 7.1% and 55.0% +/- 6.3% (t = 8.30, P < 0.01). CD133+ cells exist in a small proportion in Hep-2 cell line and they show the properties of cancer stem cells, with the resistance to irradiation and chemotherapy.

Influencing of hep-2 cell function by RNAi silencing E-cadherin expression

To explore the function of human laryngeal carcinoma Hep-2 cell after down-regulating the expression of E-cadherin gene to provide theoretical rationales for gene therapy of laryngeal cancer. According to the GenBank database, 3 pairs of shRNA sequences of E-cadherin gene were designed and synthesized. shRNAs were transfected into the cell line Hep-2 by liposome. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the silencing effect of E-cadherin expression. The changed capacity of cell proliferation were detected in vitro by methyl thiazolyl tetrazolium (MTT) assay in the transfected Hep-2 cells and the cell proliferation rate (survival rate) was calculated. And Transwell was used to detect the migratory capacity of Hep-2 cells after siRNA transfection. The E-cadherin gene expression of RNAi transfected Hep-2 cells significantly decreased in interference group. And the proliferation of interference group became markedly enhanced. In Transwell test, the migrated cell numbers in interference group were significant higher than those in negative control group (262 ± 15, 288 ± 12, 292 ± 6 vs 74 ± 8, all P < 0.01). RNA interference silencing of E-cadherin gene expression can significantly enhance the proliferation and migratory capacity of Hep-2 cells. And E-cadherin may be considered as one of gene therapy targets for laryngeal cancer.

S100A11 Is a Migration-Related Protein in Laryngeal Squamous Cell Carcinoma

Objective: As a member of the S100 proteins family, the involvement of S100A11 has been suggested in a wide range of biological processes such as cell growth and motility, cell-cycle progression, transcription, differentiation and smooth muscle cell migration. However, the expression of S100A11 and its exact function in laryngeal squamous cell carcinoma (LSCC) have not been elucidated.Methods: The protein and mRNA expression levels of S100A11 were analyzed in primary tumors and matched tumor-adjacent tissues of LSCC by western blotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) or quantitative real time PCR (Q-RT-PCR), respectively. Cell proliferation, colony formation, migration and wound-healing assays were performed to assess whether the knockdown of S100A11 by small interfering RNA (siRNA) could influence the biological behavior of human laryngeal carcinoma Hep-2 cells in vitro.Results: We found that both protein and mRNA levels of S100A11 were overexpressed in laryngeal tumor tissues when compared to the corresponding noncancerous tissues. Further, it was demonstrated that the expression of S100A11 could induce migration but not proliferation of Hep-2 cells. Additionally, S100A11 altered a series of intracellular events, including the down-regulation of epidermal growth factor receptor (EGFR), CD44 and MMP2.Conclusions: These results highlight the significance of S100A11 in LSCC progression and suggest that the dysregulation of S100A11 might contribute to the metastatic progression of LSCC.

Overexpression, purification, and pharmacologic evaluation of anticancer activity of ribosomal protein L24 from the giant panda (Ailuropoda melanoleuca)

The ribosomal protein L24 (RPL24) belongs to the L24E family of ribosomal proteins and is located in the cytoplasm. The purpose of this study was to investigate the structure and anti-cancer function of RPL24 of the giant panda (Ailuropoda melanoleuca). The complementary DNA of RPL24 was cloned successfully using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing RPL24 complementary DNA and overexpressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified using Ni-chelating affinity chromatography. The results indicated that the length of the fragment cloned is 509 bp, and it contains an open-reading frame of 474 bp encoding 157 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL24 protein is 17.78 kDa with a theoretical isoelectric point of 11.86. The RPL24 gene is readily expressed in E. coli, and the RPL24 fused with the N-terminal histidine-tagged protein to give rise to the accumulation of an expected 23.51-kDa polypeptide. The inhibitory rate in mice treated with 0.1 mg/mL RPL24, the highest of 3 doses administered, can reach 67.662%, which may be comparable to the response to mannatide. The histology of organs with tumors showed that the tissues in the RPL24 group displayed a looser arrangement compared with that in the control group. Furthermore, no obvious damage was apparent in other organs, such as heart, lung, and kidney. The data showed that the recombinant RPL24 had time and dose dependency on the cell growth inhibition rate. Human laryngeal carcinoma Hep-2 cells treated with 0.3125-10 µg/mL RPL24 for 24 h displayed significant cell growth inhibition (P < 0.05; N = 6) in assays using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide compared with that in control (untreated) cells. By contrast, human hepatoma Hep G-2 cells displayed no significant change (P > 0.05; N = 6) from control (untreated) cells. RPL24 has time and dose dependency on Hep-2 cell growth inhibition. The data indicate that the effect at low concentrations is better than that at high concentrations, and the concentration of 0.625 µg/mL provides the best rate of growth inhibition. Further research is ongoing to determine the bioactive principles of recombinant RPL24 protein that are responsible for its anticancer activity.

Iron-Cobalt Ferrite Nanoparticles—Biocompatibility and Distribution After Intravenous Administration to Rat

The biocompatibility of iron-cobalt ferrite magnetic nanoparticles (MNP) in vitro and in vivo and their distribution under an external magnetic field after intravenous administration to tumor-bearing rats were studied. In vitro rat mesenchymal stem cells and human larynx carcinoma HEp-2 cells accumulated the magnetic material intracellularly. The proliferation of HEp-2 cells was inhibited moderately by the MNP containing ca. 1.5% Co (either uncoated or polysiloxane-coated) and effectively-by the MNP containing ca. 25% Co. In vivo the most MNP-accumulating organs were the spleen, the kidneys, and the liver. No accumulation of the MNP was observed in the brain. It was found that the differences in the MNP distribution caused by topical application of a magnet were more pronounced at 24 h post injection than at 0.5-3.0 h-while the expected plasma life of the MNP doesn't exceed 10 min. E.g., application of the magnet caused a 2.4-fold MNP-attributed increase in iron concentration in M-1 sarcoma model at 24 h post injection. Similarities were found in lymph tissue tropism between our particles that readily agglutinate in plasma-containing media and nonagglutinating, long-circulating MNP reported by other authors.

Redox regulation of calcium signaling in cancer cells by ascorbic Acid involving the mitochondrial electron transport chain.

Previously, we have reported that ascorbic acid regulates calcium signaling in human larynx carcinoma HEp-2 cells. To evaluate the precise mechanism of Ca2+ release by ascorbic acid, the effects of specific inhibitors of the electron transport chain components on mitochondrial reactive oxygen species (ROS) production and Ca2+ mobilization in HEp-2 cells were investigated. It was revealed that the mitochondrial complex III inhibitor (antimycin A) amplifies ascorbate-induced Ca2+ release from intracellular stores. The mitochondrial complex I inhibitor (rotenone) decreases Ca2+ release from intracellular stores in HEp-2 cells caused by ascorbic acid and antimycin A. In the presence of rotenone, antimycin A stimulates ROS production by mitochondria. Ascorbate-induced Ca2+ release in HEp-2 cells is shown to be unaffected by catalase. The results obtained suggest that Ca2+ release in HEp-2 cells caused by ascorbic acid is associated with induced mitochondrial ROS production. The data obtained are in line with the concept of redox signaling that explains oxidant action by compartmentalization of ROS production and oxidant targets.

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca)

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.

Inhibitory effect of Jab1-specific shRNA on the progression of human laryngeal carcinoma xenografts nude mice

To evaluate the antitumour efficacy of shRNA plasmid specifically targeting Jab1 gene. The nude mouse tumor model was made by subcutaneous injection of human laryngeal carcinoma Hep-2 cells. The tumor growth was monitored after intratumoral injection of pJab1, pKB plasmids and saline. Jab1 and p27 expressions in tumour tissues were examined by immunohistochemistry staining and RT-PCR. Mean volume of the pJab1-treated tumors was (267.60 ± 88.19) mm(3), significantly less than that of tumors treated with pKB plasmids (832.20 ± 140.39) mm(3) or saline (895.40 ± 145.93) mm(3) (F = 36.73, P < 0.001). Immunohistochemistry showed that the expression of Jab1 protein was significantly reduced in the pJab1-treated group (32.40% ± 5.59%) compared to the control groups, whereas the expression rate of p27 protein in the pJab1 group (76.80% ± 6.30%) was significantly increased compared to the control groups (P < 0.001). The down regulation of Jab1 protein by pJab1 plasmid was consistent with mRNA expression confirmed by RT-PCR. The level of Jab1 mRNA level in the pJab1-treated group (0.65 ± 0.03) was significantly lower than the control groups (F = 558.00, P < 0.001), however, p27 mRNA, was 0.80 ± 0.02, had no significant alteration (F = 1.52,P > 0.05). The pJab1 plasmid results in downregulation of Jab1 in an xenograft tumour model of human laryngeal carcinoma Hep-2 cells and significantly inhibits the tumour growth in vivo. This suggests that pJab1 plasmid specifically targeting Jab1 gene expression could be an effective therapy for human laryngeal carcinoma.

Antitumor potential of SLPI promoter controlled recombinant caspase-3 expression in laryngeal carcinoma

The purpose of this study is to develop a specific and efficient targeted gene therapy candidate approach for laryngeal carcinomas. Several promoters of human squamous cell carcinoma antigen 2(SCCA2), secretory leukocyte protease inhibitor (SLPI) and Survivin genes were cloned from human genomic DNA and evaluated for tumor-specific transcription potential in human laryngeal carcinoma Hep-2 cells by dual luciferase assays. One SLPI promoter fragment (677 bp) showed the highest efficiency and specificity, and was used to control the expression of a recombinant active caspases-3 (revCasp3), which could trigger apoptosis without activation of its upstream cascade elements once expressed in a cell, in an adenoviral vector (Ad-SLPI-revCasp3), and its antitumor efficacy was assessed. In vitro infection with Ad-SLPI-revCasp3 showed revCasp3 could be specifically expressed in Hep-2 cells, resulting in efficient activation of endogenous Caspase-3 and subsequent apoptosis of Hep-2 cells. In Hep-2 nude mice xenograft model, intratumoral administration of Ad-SLPI-revCasp3 significantly inhibited tumor growth without obvious loss of body weight and obvious hepatic toxicity. In summary, our study showed the specific and efficient apoptosis-inducing potential of Ad-SLPI-revCasp3, and this makes it a new candidate approach of targeted gene therapy for laryngeal squamous cell carcinoma, which needs further systematic investigation.

Targeted Inhibition of Mammalian Target of Rapamycin (mTOR) Signaling Pathway Inhibits Proliferation and Induces Apoptosis of Laryngeal Carcinoma Cells in vitro

Laryngeal carcinoma is one of the most aggressive cancers of the head and neck region. The survival rate of patients with laryngeal carcinoma is low due to its late metastases and resistance to chemotherapy and radiotherapy. It was reported that mTOR was involved in the growth and apoptosis of various cancer cells. The aim of this study was to detect the effects of mTOR inhibition by mTOR shRNA on the proliferation, apoptosis and invasive ability of Hep-2 human laryngeal carcinoma cells in vitro. mTOR shRNA was designed and transfected into Hep-2 human laryngeal carcinoma cells. Untreated cells and cells treated with control vector (non-targeted shRNA) were used as control. The proliferation and apoptosis of Hep-2 cells were detected by MTT and flow cytometry. A transwell assay was used to measure the invasive ability of Hep-2. The inhibition effects on the mTOR signaling pathway by mTOR shRNA were studied using RT-PCR and Western blot. Our results showed that the mRNA and protein expression of mTOR and Akt were high in laryngeal carcinoma cells and could be inhibited by mTOR shRNA. At the same time, low expression of PTEN mRNA and protein was observed in Hep-2 cells. The expression increased when the cells were transfected with mTOR shRNA. This showed that mTOR shRNA could inhibit the proliferation and invasive ability of Hep-2 cells. It also could induce the apoptosis of Hep-2 cells in vitro. The mTOR signaling pathway plays an important role in the development of laryngeal carcinoma. The mTOR shRNA we designed in this experiment effectively inhibited the mTOR signaling pathway. It inhibited the proliferation and invasive ability of the studied laryngeal carcinoma cells and induced their apoptosis in vitro. mTOR might therefore be a useful target in the therapy of laryngeal carcinoma.

尼美舒利对人喉鳞癌Hep-2细胞祼鼠移植瘤CD44和MMP-7表达的影响

Objective To evaluate the inhibitory effect of Nimesulide on tumor growth of human laryngeal squamous carcinoma in nude mice and to explore the possible mechanism involved in the inhibition.Methods Nude mice model bearing human laryngeal squamous carcinoma cell line(Hep-2)was established.The volumes of the subcutaneous tumor were observed after Nimesulide treatment.The levels of COX-2,CD44 and MMP-7 protein and mRNA in transplantation tumor were examined by immunohistochemistry and RT-PCR,respectively.Results The growth of the carcinoma transplanted was significantly inhibited in the treatment group.Compared with the control group,the Nimesulide treatment group had significantly reduced weight and volume of tumor(all P0.05).There was no significantly difference in weights of mice increasing between the two groups.The levels of COX-2,CD44 and MMP-7 protein and mRNA of the treatment group were lower than those of the control group(all P0.05).Conclusion NIM has obvious inhibitory effect on the growth of human laryngeal squamous cell carcinoma Hep-2 cell xenografts in nude mice,through the down-regulation COX-2,CD44 and MMP-7.

Promoting effect of Chlamydia pneumoniae infection on human laryngeal carcinoma HEp-2 cell adhesion and migration

To explore the effect of Chlamydia pneumoniae (C.pn) infection on human laryngeal carcinoma cell line HEp-2 cell adhesion and migration, to further clarify the role and mechanism of C.pn infection in tumor metastasis. HEp-2 cells were infected with C.pn after the culture and propagation of C.pn. The cytopathic effect was observed by microscopy. Morphological characteristics of C.pn inclusions in HEp-2 cells were examined by fluorescence microscopy and acridine orange staining. The ultrastructural changes of C.pn inclusions in the HEp-2 cells were examined by transmission electron microscopy (TEM). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of HEp-2 cells to collagen I. Wound-healing assay and transwell assay were performed to explore the effect of C.pn infection on HEp-2 cell migration. At 72 h post-infection, C.pn infected-HEp-2 cells were swollen and partially desquamated. Numerous vacuoles (inclusions) were observed and C.pn inclusions occupied almost the whole cytoplasm of the HEp-2 cells. Grape-like C.pn inclusions were observed in the HEp-2 cells stained with acridine orange under a fluorescence microscope at 72 h after infection. Under TEM, there were more mature pear-shaped elementary bodies, but less larger and round reticulate bodies in the HEp-2 cells infected with C.pn for 72 h. In the cell adhesion assay, the A value in C.pn infection group was 0.669 ± 0.011, significantly higher than that in the control group (0.558 ± 0.005) at 2 h after infection (P < 0.001). The cell adhesion ratio in the C.pn infection group was 119.89%. The migration distance of C.pn infected-HEp-2 cells in the wound-healing assay was significantly longer than that of control cells at 24 h after infection (P < 0.05). HEp-2 cells infected with C.pn for 12 h migrated more than the control cells in the transwell assay (23.40 ± 2.41 vs 10.40 ± 1.67) (P < 0.001). C.pn infection can significantly promote HEp-2 cell adhesion to collagen I and migration of HEp-2 cells, indicating that C.pn infection may play an important role in promoting the metastasis of laryngeal cancer.

Rapid functional screening of effective siRNAs against Plk1 and its growth inhibitory effects in laryngeal carcinoma cells

Plk 1 is overexpressed in many human malignancies including laryngeal carcinoma. However, its therapeutic potential has been never examined in laryngeal carcinoma. In the present study, a simple cellular morphology-based strategy was firstly proposed for rapidly screening the effective siRNAs against Plk1. Furthermore, we investigated the effects of Plk1 depletion via a novel identified effective siRNA against Plk1, Plk1 siRNA-607, on human laryngeal carcinoma Hep-2 cells. The results indicated that Plk1 siRNA-607 transfection resulted in a significant inhibition in Plk1 expression in cells, and subsequently caused a dramatic mitotic cell cycle arrest followed by massive apoptotic cell death, and eventually resulted in a significant decrease in growth and viability of the laryngeal carcinoma cells. Taken together, our present study not only suggests a simple strategy for rapidly screening effective siRNAs against Plk1 but also implicates that Plk1 may serve as a potential therapeutic target in human laryngeal carcinoma.

Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin

Earlier, we have shown that spontaneously isolated non-pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin-like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep-2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli-carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep-2 cells. Moreover, internalization of non-invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.

Effects of Epigallocatechin gallate glucoside on antitumor activities in human laryngeal epidermoid carcinoma Hep2

(−)-Epigallocatechin-3-gallate (EGCG) has been reported to inhibit cellular proliferation and induce apoptosis in a range of cancer cells. This study examined the cytotoxicity of EGCG glucoside on human laryngeal epidermoid carcinoma Hep2 cells. The EGCG glucoside treatment decreased the cell viability in Hep2 cells. Furthermore, EGCG glucoside caused apoptotic morphological changes with chromatin condensation and nuclear fragmentation, as observed by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and activated caspase-3 immunohistochemisty. However, the EGCG glucoside did not induce the production of reactive oxygen species (ROS), suggesting that oxidative stress is not involved in the apoptotic response. EGCG glucoside induces apoptosis in Hep2 cells through not the generation of ROS, but the activation of caspase-3.

Induction of G2/M Phase Arrest and Apoptosis by Oridonin in Human Laryngeal Carcinoma Cells

Oridonin (1), an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects. In this study, the mechanism involved in 1-induced growth inhibition, including apoptosis and G(2)/M phase arrest, in human laryngeal carcinoma HEp-2 cells deficient in functional p53, was investigated for the first time. Compound 1 triggered the mitochondrial apoptotic pathway, as indicated by increased Bax/Bcl-2 ratios, reduction of mitochondrial membrane potential (DeltaPsi(m)), and substantial increase in apoptosis-inducing factor (AIF) and cytochrome c. Inhibition of caspase-9 in HEp-2 cells did not protect the cells from 1-induced apoptosis, and cleaved caspase-9 was not detected, indicating that apoptosis occurred via a caspase-9-independent pathway. The results also suggested that G(2)/M phase arrest and apoptosis mediated by 1 occurred via a p53-independent but in a p21/WAF1-dependent manner in HEp-2 cells. In addition, the generation of reactive oxygen species (ROS) was found to be a critical mediator in growth inhibition induced by 1. Taken together, the results indicate that oridonin (1) is a potentially effective agent for the treatment of laryngeal squamous cell carcinoma.

RNA interference targeting c-Met inhibits proliferation of human laryngeal carcinoma Hep-2 cell line in vitro and in its xenografts in nude mice

The proto-oncogene c-Met was found to express on human laryngeal carcinoma Hep-2 cell line in previous research. In the present study, the author further examined whether inhibition of c-Met by RNA interference (RNAi) might inhibit biologic activity of Hep-2 cell line in vitro and proliferation using a murine laryngeal carcinoma model. RNAi plasmid that can express small interfering RNA targeting c-Met or siRNA that did not match any known human coding mRNA(control siRNA plasmid)was designed, constructed, and transfected into Hep-2 cell line by using cationic liposome Lipofectamine2000 as transfecting agent. In vitro, the transfection efficacy was tested by RT-PCR and Western Blot method, then elected the most inhibitive c-Met-siRNA sequence. Cell proliferation, movement and invasion were studied using MTT, cell migration assay and cell invasion assay, respectively. The Hep-2 cells were transplanted into nude mice, then the time of tumor formation and growth were observed. After tumor formation, c-Met-siRNA was given as the anti-tumor therapy. Expression of c-Met, MMP-9 and VEGF were detected by Western Blot method. After the pSilencer2.0/c-Met-shRNA recombinant plasmid transfection into laryngeal carcinoma Hep-2 cells, the expression of mRNA and protein of c-Met decreased significantly in Hep-2 cells. On the 35th day after tumor vaccination, the tumor volume was (138 ± 27) mm³ in c-Met-siRNA transfection group, Which was diminished significantly in contrast with control group (P < 0.01). The expression of c-Met, MMP-9 and VEGF in the tumor of experiment group was decreased significantly, respectively (P < 0.05). The results indicated that c-Met-siRNA can down-regulate the expression of c-Met and markedly inhibit laryngeal carcinoma Hep-2 cell proliferation, movement and invasion and the growth of transplantation tumor of nude mice. The siRNA expressing plasmid mediated gene therapy might be a new strategy in targeting molecular therapy of cancer of larynx.