RESUMEN
Granzyme B and perforin, two of the most important components, have shown anticancer properties in various cancers, but their effects in laryngeal cancer remain unexplored. Here we decided to examine the effects of Granzyme B and perforin in Hep-2 cells and clarify the role of perforin and granzyme B in the tumorigenicity of laryngeal cancer cell line. Hep-2 cells were transfected with pVAX1-PIG co-expression vector (comprising perforin and granzyme B genes), and then the growth and apoptosis of these Hep-2 cells were evaluated. The tumorigenicity of Hep-2 cell line co-expressing perforin and granzyme B genes was tested in BALB/c nu/nu mice. We found that the co-expression of perforin and granzyme B genes could obviously inhibit cell focus formation and induce cell apoptosis in Hep-2 cells. Furthermore, after subcutaneous injection of Hep-2 cells transfected with pVAX1-PIG, an extensive delay in tumor growth was observed in BALB/c-nu/nu mice. Moreover, our studies demonstrated that the anticancer activity of perforin and granzyme B was sustainable in vivo as tumor development by inducing cell apoptosis. Taken together, our data indicate that the co-expression of perforin and granzyme B genes exhibits anticancer potential, and hopefully provide potential therapeutic applications in laryngeal cancer.
Asunto(s)
Apoptosis/genética , Carcinogénesis/metabolismo , Granzimas/biosíntesis , Neoplasias Laríngeas/metabolismo , Perforina/biosíntesis , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Citometría de Flujo , Granzimas/genética , Xenoinjertos , Humanos , Inmunohistoquímica , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Electrónica de Transmisión , Perforina/genética , TransfecciónRESUMEN
OBJECTIVE: To establish human bronchial epithelial cell lines over expressing oncogene and to investigate its application in detection of carcinogen-induced cell transformation. METHODS: Mediated by retrovirus infection, human telomerase catalytic subunit, hTERT was introduced into immortal human bronchial epithelial cells (16HBE) and followed by introduction of the oncogenic allele H-Ras(V12), or c-Myc or empty vector, creating cell lines 16HBETR, 16HBETM and 16HBETV, respectively. Biological characteristics of these cell lines including morphology, proliferation, and chromosomal aberration were examined to access whether they were transformed. Soft agar experiment and nude mice subcutaneous injection were performed using pre-transformed 16HBE cells induced by known carcinogens, nickel sulfate (NiSO4) and 7, 8, -dihydrodiol-9, 10-epoxide benzo[a] pyrene (BPDE). RESULTS: With detection of telomerase activity and Western blotting, the expression of target proteins was verified. Thus, the transgenic 16HBE cell lines were successfully established. Cells expressing oncogene H-Ras or c-Myc grew 30.3% or 10.4% faster than control cells. However, these cells failed to form colonies in soft agar or form tumor in nude mice. 16HBETR, 16HBETM cells obtained transformed phenotype at 5 wks, 11 wks, respectively after treatment with BPDE, which are 15 wks and 9 wks earlier than control cells 16HBETV (20 wks). Meanwhile, 16HBETR, 16HBETM cells obtained transformed phenotype at 11 wks, 14 wks, respectively after treatment with nickel sulfate, which are 21 wks and 18 wks earlier than control cells (32 wks). CONCLUSION: With the advantage of shorter latency, transgenic human cell transformation models could be used in potent carcinogen screening and applied to chemical-carcinogenesis mechanism study.
Asunto(s)
Línea Celular , Transformación Celular Neoplásica , Células Epiteliales , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Animales , Pruebas de Carcinogenicidad , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Expresión Génica , Regulación de la Expresión Génica , Genes myc , Genes ras , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
OBJECTIVE: To evaluate and screen the specific RNAi fragments which can effectively inhibit Aryl hydrocarbon receptor(AHR) gene mRNA expression in human bronchial epithelial cell line (16HBE). METHODS: AHR mRNA of 16HBE cells transfected 4 different AHR gene interfere sites were determined quantitatively with the quantitative competitive RT-PCR by using self-prepared internal standard as competitive templates, and the RNA interfere effect wasevaluated. RESULTS: AHR mRNA average expression per 40ng total RNA of 16HBE cells transfected 4 different AHR gene interfere fragments were 5.65fg, 14.78fg, 3.14fg and 0.68fg respectively, the average rates of inhibition were 61.6%, -0.5%, 78.6% and 95.4% respectively. CONCLUSION: AHR gene specific effective RNA interfere sequence ware screened by quantitative competitive RT-PCR which could accurately quantify gene mRNA level, and offered condition for studying the gene function of AHR.
Asunto(s)
Bronquios/citología , Células Epiteliales/metabolismo , ARN Interferente Pequeño/genética , Receptores de Hidrocarburo de Aril/genética , Unión Competitiva , Línea Celular , Células Epiteliales/citología , Humanos , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
BACKGROUND & OBJECTIVE: Carcinoembryonic antigen (CEA) positive cancers are poorly responded to different kinds of treatments. Gene vaccines are promising in research of gene immunotherapy for these tumors. In this study, a CEA gene vaccine with hIL-2 as an immune adjuvant was constructed into a pVAX1 vector for synchronous expression, so as to explore experimentally a new biotherapy strategy against tumors. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), CEA cDNA was obtained from a large intestine carcinoma tissue; its encoded protein was compared with the CEA presented in GenBank using the protein analysis software. The acquired CEA cDNA fragment was linked to hIL-2 cDNA via an IRES site and cloned into the pVAX1 vector. The recombinant plasmid was estimated by CEA luminometry assay and hIL-2 ELISA measurement respectively. RESULTS: The nucleotide sequences of the target gene fragments of the recombinant plasmid were verified. The acquired CEA sequence is highly homologous with M29540 and M17303 (99.8%) in GenBank; and the PCR sequence of hIL-2 is coincident with the original cDNA (100%) provided. The antigenicity,membrane-spanning segments, signal cleavage sites, secondary structure and 3D structure of the acquired CEA protein were similar to the original proteins of M29540 and M17303 predicted by the protein analysis software. Results showed the recombinant could steadily express CEA antigen and hIL-2 protein synchronously in CHO cells in vitro. CONCLUSION: The CEA cDNA was obtained from the tumor tissue and the CEA gene vaccine with hIL-2 coexpression was constructed successfully. It has provided a possible method for immunotherapy against CEA positive cancers in vivo.