RESUMEN
Cholangiocarcinoma (CCA) is the most common type of malignant tumor of the bile duct and is characterized by high morbidity and mortality; it is difficult to diagnose in the early stages and responds poorly to current conventional radiotherapy and chemotherapy. The present study investigated the role of GSK3ß signaling on the anticancer effects of doxorubicin in human CCA cells. Blocking GSK3ß enhanced the sensitivity of human CCA cells to doxorubicin (Dox)induced apoptosis, which was accompanied by decreased AKT and focal adhesion kinase (FAK) activity. Moreover, inhibiting GSK3ß using 6bromoindirubin3'oxime, CHIR99021 or small interfering RNA decreased phosphorylation of FAK and AKT, and promoted apoptosis of Doxinduced human CCA cells. Moreover, FAK inhibition suppressed AKT activity independently of phosphoinositide 3kinase activity. These results indicated that GSK3ß protects human CCA cells against Doxinduced apoptosis via sustaining FAK/AKT activity.
Asunto(s)
Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Doxorrubicina/farmacología , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/genética , Humanos , Indoles/farmacología , Oximas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño/farmacologíaRESUMEN
Increasing cell mobility is the basis of tumor invasion and metastasis, and is therefore a therapeutic target for preventing the spread of many types of cancer. Septins are a family of cytoskeletal proteins with GTPase activity, and play a role in many important cellular functions, including cell migration. SEPT9 isoform 1 protein (SEPT9_i1) has been associated with breast tumor development and the enhancement of cell migration; however, the exact mechanism of how SEPT9_i1 might affect breast cancer progression remains to be elucidated. Here, we report that the expression of SEPT9_i1 positively correlated with paxillin, and both were significantly upregulated in invasive breast cancer tissues of patients with lymph node metastases. Lentivirus-mediated shRNA knockdown of SEPT9 in MCF-7 cells diminished tumor cell migration, focal adhesion (FA) maturation and the expression of ß-actin, ß-tubulin, Cdc42, RhoA, and Rac, whereas overexpression of SEPT9_i1 in SEPT9-knockdown MCF-7 cells promoted cell migration, FA maturation and relevant protein expression. Furthermore, overexpression of SEPT9_i1 in MCF-7 cells markedly increased FAK/Src/paxillin signaling, at least in part through RhoA/ROCK1 upstream activation. Transcriptome profiling suggested that SEPT9_i1 may directly affect "Focal adhesion" and "Regulation of actin cytoskeleton" signaling mechanisms. Finally, overexpression of SEPT9_i1 markedly enhanced lung metastases in vivo 6 weeks after tumor inoculation. These findings suggest that a mechanism of Septin-9-induced aberrant cancer cell migration is through cytoskeletal regulation and FA modulation, and encourages the use of SEPT9 as novel therapeutic target in the prevention of tumor metastasis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/genética , Metástasis Linfática , Septinas/metabolismo , Quinasas Asociadas a rho/metabolismo , Citoesqueleto de Actina/genética , Adulto , Animales , Neoplasias de la Mama/genética , Adhesión Celular/genética , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/patología , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA-Seq , Septinas/química , Septinas/genética , Transducción de Señal/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
OBJECTIVE: To explore the effect of combined gene therapy with interference hTERT and TRF2 gene on the treatment of breast cancer. METHODS: Recombinant adenovirus rAd-hTERT and rAd-TRF2 expressing siRNA-hTERT and siRNA-TRF2 was constructed, and the vectors were transfected into MCF-7 cells. Than the expressions of hTERT and TRF2 proteins were detected by Western blot, the inhibition of MCF-7 cell proliferation by MTT colorimetry, and the changes of MCF-7 cell cycle by flow cytometry and the colony forming ability of MCF-7 cells by clone form test. RESULTS: At 48 h after transfection, the relative expression amounts of hTERT protein of the PBS control group, rAd-blank group, rAd-HK control group, rAd-hTERT group, rAd-TRF2 group and rAd-hTERT and rAd-TRF2 group were 1.00, 0.94 +/- 0.02, 0.95 +/- 0.04, 0.18 +/- 0.04, 0.95 +/- 0.01 and 0.18 +/- 0.04, respectively. The relative expression amounts of TRF2 protein were 1.00, 1.01 +/- 0.08, 0.96 +/- 0.02, 0.95 +/- 0.08, 0.22 +/- 0.01 and 0.26 +/- 0.02, respectively. After transfection of rAd-hTERT or rAd-TRF2 into MCF-7 cells separately, the inhibition rate of cell proliferation was only 54.6% and 48.4%, there was 8.9% +/- 1.2% or 9.2% +/- 2.3% of MCF-7 cells into M phase, 66.4% +/- 1.5% or 64.6% +/- 1.9% of MCF-7 cells was arrested at G(0)/G(1) phase, and the cell colony forming ability was decreased significantly (cell colony number from 100 in PBS control group down to 41.3 +/- 5.1 and 43.7 +/- 6.4). But after transfection by rAd-hTERT and rAd-TRF2 simultaneously, the inhibition rate of cell proliferation was about 82.1%, and M phase cells was significantly reduced to 4.4% +/- 1.2%. Large numbers of cells were arrested at G(0)/G(1) phase (81.4% +/- 1.3%), and the cell colony forming ability was more significantly decreased (cell colony number there were only 29.2 +/- 3.9). CONCLUSION: More effective effect of tumor gene therapy can be achieved by combination of interference hTERT and TRF2 genes as compared with interference by either of the single gene alone.
Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular , Interferencia de ARN , Telomerasa/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Adenoviridae/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Terapia Genética , Vectores Genéticos , Humanos , ARN Interferente Pequeño/genética , Telomerasa/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
To explore the inhibitory effect in different positions siRNA on hTERT mRNA expression in MCF-7 cells at the different time points, we Designed and chemically synthesized four pairs of siRNA which aimed at human telomerase reverse transcriptase (hTERT) gene specifically, which were transfected into the MCF-7 cells by liposome method, and then the expression of hTERT mRNA in MCF-7 cells was tested respectively by half-quantity RT-PCR at 12 h, 24 h, 48 h, 72 h, and 5 d after transfection. Compared with the control groups, there were three pieces of siRNA that inhibit the expression of hTERT mRNA at 12 h after transfection among the four pieces. The highest inhibition ratio occurred at 48 h after transfection, and after 72 h the ratio descended, when the siRNA sequence was located at the relative simple structure site in the secondary structure of hTERT mRNA had the highest inhibition ratio, which was 75%(P<0.01), which indicated that the specific siRNA had obvious inhibition effect on hTERT gene, and the effect was dependant positionally and timely dependence.