Search

According to the cancer stem cell theory, a small subpopulation of cancer cells, known as cancer stem cells (CSCs), exist that are self-renewing and are involved in tumor invasion, metastasis and recurrence. A number of studies have reported that certain cancer cells are able to efflux the Hoechst 33342 dye. These cells are termed side population (SP) cells and share characteristic features of CSCs. The results of the present study revealed that 2.7% of primary head and neck squamous cell carcinoma (HNSCC) cells were SP cells. This was reduced to 0.7% following treatment with verapamil. The immunofluorescence and reverse transcription polymerase chain reaction analysis revealed that SP cells have an enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC subfamily G, member 2 (ABCG2), which has been identified to be actively involved in drug exclusion. Similarly, the mRNA level of the oncogene B lymphoma Mo-MLV insertion region-1 and the stem cell surface proteins nestin and octamer-binding transcription factor-4 were highly expressed in the SP cells compared with the non-SP cells. In addition, it was demonstrated that HNSCC SP cells exhibited increased proliferation and were highly resistant to multiple drugs. These findings suggest that the presence of CSCs, such as SP cells, may be responsible for chemotherapy failure and tumor relapse in patients with HNSCC. Therefore, the identification of a novel therapeutic drug that could effectively target CSCs may help to eradicate refractory tumors.

To determine the inhibitory effect of the synthetic STAT3 siRNA on the expression of STAT3 gene in human laryngeal cancer cell lines Hep2 and to investigate the effect of STAT3 siRNA on growth and apoptosis in Hep2 cells. A pair of DNA templates coding siRNA against STAT3-mRNA was synthesized to reconstruct plasmid of pSilencer1.0-U6 siRNA-STAT3. Hep2 cells were transfected with RPMI-1640 media (untreated), plasmid (empty), and STAT3 siRNA, respectively. Northern blot and Western blot analysis of STAT3 and pTyr-STAT3 expression in Hep2 cells and Western blot analysis of Bcl-2 expression in the Hep2 cell was performed 72 h after transfection. MTT, flow cytometry, and AO/EB assay were used for determination of cells proliferation and apoptosis in Hep2 cells. pTyr-STAT3 was markedly expressed in untreated Hep2 cells and the vector-treated Hep2 cells, whereas pTyr-STAT3 expression was significantly reduced in STAT3 siRNA-transfected Hep2 cells, indicating that STAT3 siRNA inhibited the activity of STAT3. Transfection of Hep2 cells with STAT3 siRNA significantly inhibited STAT3 expression at both mRNA and protein level in Hep2 cells and the inhibition was characterized by time-dependent transfection. Treatment of Hep2 cells with STAT3 siRNA resulted in dose-dependent growth inhibition of Hep2, this significantly increased apoptotic cell rate, and decreased Bcl-2 expression level in Hep2 cells. STAT3 siRNA had an effect on induction of either early or late stage apoptosis. This study demonstrates that STAT3 siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading to growth suppression and induction of apoptosis in Hep2 cells. The use of siRNA technique may provide a novel therapeutic approach to treat laryngeal cancer and other malignant tumors expressing constitutively activated STAT3.

The present study aimed to identify key genes and relevant microRNAs (miRNAs) involved in laryngeal squamous cell carcinoma (LSCC). The gene expression profiles of LSCC tissue samples were analyzed with various bioinformatics tools. A gene expression data set (GSE51985), including ten laryngeal squamous cell carcinoma (LSCC) tissue samples and ten adjacent non-neoplastic tissue samples, was downloaded from the Gene Expression Omnibus. Differential analysis was performed using software package limma of R. Functional enrichment analysis was applied to the differentially expressed genes (DEGs) using the Database for Annotation, Visualization and Integrated Discovery. Protein-protein interaction (PPI) networks were constructed for the protein products using information from the Search Tool for the Retrieval of Interacting Genes/Proteins. Module analysis was performed using ClusterONE (a software plugin from Cytoscape). MicroRNAs (miRNAs) regulating the DEGs were predicted using WebGestalt. A total of 461 DEGs were identified in LSCC, 297 of which were upregulated and 164 of which were downregulated. Cell cycle, proteasome and DNA replication were significantly over-represented in the upregulated genes, while the ribosome was significantly over-represented in the downregulated genes. Two PPI networks were constructed for the up- and downregulated genes. One module from the upregulated gene network was associated with protein kinase. Numerous miRNAs associated with LSCC were predicted, including miRNA (miR)-25, miR-32, miR-92 and miR-29. In conclusion, numerous key genes and pathways involved in LSCC were revealed, which may aid the advancement of current knowledge regarding the pathogenesis of LSCC. In addition, relevant miRNAs were also identified, which may represent potential biomarkers for use in the diagnosis or treatment of the disease.

Objective: To investigate the inhibitory effect of plasmid-based survivin-specific short hairpin RNA and GRIM-19 on the growth of Hep-2 laryngeal cancer cells. Methods: The plasmid expressing survivin-specific short hairpin RNA (shRNA) and GRIM-19 (p-siRNA survivin/GRIM-19) was prepared and transfected into Hep-2 cells with Lipofectamine 2000. The mRNA and protein expression of surviving and GRIM-19 were measured with RT-PCR and western blot assay, respectively. MTT assay was employed to detect the proliferation of Hep-2 cells, and flow cytometry and AO/EB assay were done to determine the apoptosis of Hep-2 cells. Results: In the p-siRNA survivin/GRIM-19, the mRNA and protein expression of survivin was markedly reduced by 54.4% and 42.2%, and the reduction in protein expression of surviving was more obvious than that in the p-siRNA survivin group (37%) (P

To study the effect of silencing survivin on the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo.Hep-2 cells were transfected with pGCsilencer-siRNA-survivin (psi-survivin)by Lipofectamine 2000. The mRNA and protein expressions of survivin were detected by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation activity was measured by MTT assay. Apoptosis was assessed by flow cytometry. The implanted tumors were formed from injected Hep-2 cells in nude mice. After the tumor formation, psi-survivin was injected into peritumor tissues. The growth of tumor were observed. The tumor volume was calculated and the tumor growth curve was plotted. The expression of survivin in tumor tissues was detected by Western blot. The tumor cell apoptosis was observed by Tunel staining.The sequence-specific siRNA of survivin inhibited the expressions of survivin mRNA and protein. The inhibition rates of survivin mRNA and protein expression were 54.4% and 37.0% respectively. Also the growth of Hep-2 cells was inhibited significantly, with a decrease by 71.7%. By the day 32 of tumor growth, the mean tumor volumes were (1443.9 ± 230.5) mm(3) (x(-) ± s) in saline control group, (1348.5 ± 198.4) mm(3) in plasmid-negative control group, and (624.6 ± 188.4) mm(3) in psi-survivin group, respectively (t = -5.917, P0.01). In the implanted tumors injected with psi-survivin, survivin protein expression was down-regulated significantly, with a inhibition rate of 41.8%. Tunel staining showed the apoptosis occurred in the implanted tumors.Silencing survivin could significantly inhibit the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo.

To separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells.Human laryngeal carcinoma cells were obtained by primary tissue culture technique. CD44 and CD133 molecules were used as markers to isolate the CD44(+), CD133(+), CD44(+)CD133(+) and CD44(+)CD133(-) cell subpopulations from the laryngeal carcinoma cells by flow cytometry. A nude mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. 1 x 10(6), 1 x 10(5), 1 x 10(4) and 1 x 10(3) cells were injected into the left flank of the mice, respectively. The mice were observed for palpable tumor formation and were sacrificed at 4 weeks later to assess the tumor formation rate, tumor volume and tumor weight. Boyden chamber migration assay was used to determine the migration ability and immunochemistry was used to detect the expression of stem cell antigen SCA-1 and beta1-integrin. Semi-quantities RT-PCR and Western blot analysis were performed to detect the expression level of Bmi-1 in the different cell subpopulations.The growth of subcutaneous tumors in nude mice showed that a tumor can be generated with 1 x 10(3) CD44(+)CD133(+) cells. When the same dose of 1 x 10(6) CD44(+)CD133(+) cells was injected into the mice, both the average weight and volume of the tumors were significantly higher than those generated from other cell subpopulations. Boyden chamber migration assay showed that the invasion ability of CD44(+)CD133(+) cells was significantly higher than that of other cell subsets. The results of immunochemical analysis showed an abundant expression of stem cell antigen SCA-1 and beta1-integrin in the CD44(+)CD133(+) cells. Semi-quantitative RT-PCR and Western blot analysis provided strong evidence that the Bmi-1 expression in CD44(+)CD133(+) and CD133(+) cells was very significantly higher than that in CD44(+), CD44(+)CD133(-) and control cells (P0.01).Our findings demonstrate that CD44(+)CD133(+) subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells, which may be the original cells of laryngeal carcinoma and may become a new target of tumor therapy.

Aim: To study the effect of pSilencer 1.0-U6-siRNA-stat3 on the growth of human laryngeal tumors in nude mice. Methods: Hep2 cells were transplanted into nude mice, then at the time of tumor formation, growth rates were observed. After the tumor formed, pSilencer 1.0-U6-siRNA- stat3 was injected. Tumor volumes were calculated, and growth curves were plotted. Representative histological sections were taken from mice bearing transplantation tumors in both treated and control groups, and start3 , pTyr-stat3 , Bcl-2 , cyclin D1 , and surviving expression were detected by Western blotting. survivin mRNA levels were detected by Northern blotting, hematoxylin and eosin staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay to confirm the apoptosis of tumors. Results: In nude mice, pSilencer 1.0-U6-siRNA-stat3 significantly suppressed the growth of tumors compared with controls ( P BcL2 , cyclin D1 , and surviving expression within the tumor. This significantly induced apoptosis of the tumors. Conclusion: pSilencer 1.0-U6-siRNA- stat3 was able to inhibit the growth of transplanted human laryngeal tumors in nude mice and induce apoptosis.

Nucleic vaccine of pVVP3IL-18HN expressing apoptin gene, Newcastle disease virus HN gene and IL-18 gene were constructed to observe the combinative antitumor effect of the above three genes.Eukaryotic expression plasmid pVVP3IL-18HN was constructed by inserting apoptin gene and fragment comprising fused IL-18HN gene and IRES promoter into the downstream of CMV promoter of vector pVAX1. The expression of inserted gene was identified by RT-PCR, indirect immunofluorescence and Western-blot. The recombinant plasmid was introduced into Hep-2 cells by liposome, then suppression rate of Hep-2 of different time and different quantity was calculated according to MTT results.The recombinant plasmid of pVVP3IL-18HN suppressed Hep-2 successfully and its suppression rate was up to 61.9% with 20 microg/ml, incubation of 72 hours.The nucleic vaccine constructed pVVP3IL-18HN had antitumor effect on Hep-2. It may can be used to the therapy and research of laryngeal carcinoma.