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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<0.05). The protein expression of GRIM-19 was markedly enhanced when compared with the control group (P<0.01). MTT assay revealed the proliferation of Hep-2 cells undergoing transfection with p-siRNA survivin/GRIM-19 was markedly inhibited, and the inhibition rate was as high as 79%, which was higher than that in the psi-survivin group (45%) and p-GRIM-19 group (35%). AO/EB assay and flow cytometry indicated that the apoptotic cells in the p-siRNA survivin/GRIM-19 group were dramatically increased as compared to the psi-survivin group and p-GRIM-19 group., Conclusion: The p-siRNA survivin/GRIM-19 has marked decrease in survivin expression and dramatic increase in GRIM-19 expression. Moreover, silencing of survivin and over-expression of GRIM-19 can significantly inhibit the growth and induce the apoptosis of Hep-2 in vitro and in vivo.

Background: Mounting evidence suggests that tumors are histologically heterogeneous and are maintained by a small population of tumor cells termed cancer stem cells. CD133 has been identified as a candidate marker of cancer stem cells in laryngeal carcinoma. This study aimed to analyze the chemoresistance of CD133(+) cancer stem cells., Methods: The response of Hep-2 cells to different chemotherapeutic agents was investigated and the expression of CD133 was studied. Fluorescence-activated cell sorting analysis was used to identify CD133, and the CD133(+) subset of cells was separated and analyzed in colony formation assays, cell invasion assays, chemotherapy resistance studies, and analyzed for the expression of the drug resistance gene ABCG2., Results: About 1% - 2% of Hep-2 cells were CD133(+) cells, and the CD133(+) proportion was enriched by chemotherapy. CD133(+) cancer stem cells exhibited higher potential for clonogenicity and invasion, and were more resistant to chemotherapy. This resistance was correlated with higher expression of ABCG2., Conclusions: This study suggested that CD133(+) cancer stem cells are more resistant to chemotherapy. The expression of ABCG2 could be partially responsible for this. Targeting this small population of CD133(+) cancer stem cells could be a strategy to develop more effective treatments for laryngeal carcinoma.

Objective: To study the effect of silencing survivin on the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo., Methods: 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., Results: 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, P < 0.01). 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., Conclusion: Silencing survivin could significantly inhibit the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo.

Objective: To separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells., Methods: 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., Results: 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 (P < 0.01)., Conclusion: 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.

Objective: To study the biological characteristics of CD(44)(+) stem cells in human laryngeal carcinoma., Methods: Tumor samples were obtained from 5 patients, and then the human laryngeal carcinoma cells were cultured in vitro by primary tissue culture technique. Taking CD44 molecule as a marker to isolate CD(44)(+) subpopulation cells from laryngeal carcinoma cells for further study. CD(44)(+) and CD(44)(-) cells were cultured and observed fex their development. CD(44)(+) and CD(44)(-) cells were compared in their functional status (mRNA), cell cycles (G0/G1), their degree of differentiation (CK14 and Involucrin expression) and their morphologic character of the clone., Results: The percentages of CD(44)(+) cells were about 49.8% approximately 53.5% and the median was 51.3%. After culturing CD(44)(+) cells isolated from laryngeal carcinoma could proliferate and the percentage of CD(44)(+) remained the same. CD(44)(+) tumor cells contained much less RNA, more G0/G1 cells, expressed more CK14 protein and less Involucrin protein (less differentiated state). CD(44)(+) cells were multangular in shape with protuberances; CD(44)(-) cells showed a sharp and spindle feature. In comparison with CD(44)(-) cells, CD(44)(+) cells could create heterogeneous offspring by single cell culture of limiting dilution. By observing clone forming rate after single cell planting, it was found that the CD(44)(+) cells had stronger proliferation ability., Conclusions: CD(44)(+) cells possess some characteristics of stem cells, laryngeal carcinoma stem cells maybe exist in CD(44)(+) cells.

Objective: 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., Methods: 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., Result: 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., Conclusion: 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.