RESUMO
Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.
Assuntos
Angiopoietinas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/secundário , Espermidina/análogos & derivados , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Neoplasias da Mama/irrigação sanguínea , Hipóxia Celular , Linhagem Celular Tumoral , Feminino , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Transplante de Neoplasias , Interferência de RNA , Espermidina/metabolismoRESUMO
We have developed a hypoxia-inducible gene therapy approach for the expression of the mature form of human endonuclease G to facilitate cell death in hypoxic regions of the tumor. The chimeric therapeutic gene is placed under the control of a hypoxia response element based promoter and contains a translocation motif linked in frame to an oxygen-dependent degradation domain and the endonuclease G gene. Transient expression of the chimeric therapeutic gene in breast and prostate cancer cell lines resulted in efficient cell death under hypoxia-mimetic conditions. Stable MDA-MB-435 cells expressing the chimeric therapeutic gene under 1% O2 showed an increase in stable HIF-1alpha protein levels and synthesis of the endonuclease G protein in a time-dependent manner. In normoxic conditions, these stable transgenic cells exhibited no change in growth rate, invasion and motility when compared to parental cells. Moreover, xenografts generated using the transgenic cells exhibited highly significant suppression of tumor growth in a preclinical cancer model compared to the parental cell line. Thus, the hypoxia-modulated endonuclease G expression has the potential to be used as a gene-based-therapy system to kill malignant cells within hypoxic regions of tumors.
Assuntos
Endodesoxirribonucleases/metabolismo , Terapia Genética/métodos , Neoplasias/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cobalto/farmacologia , Endodesoxirribonucleases/genética , Feminino , Humanos , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Neoplasias/genética , Neoplasias/patologia , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Carga TumoralRESUMO
Lubrol-solubilized rabbit thrombomodulin has been examined by equilibrium sedimentation in buffers that include sufficient D2O to make the detergent neutrally buoyant. Data were acquired at rotor speeds from 12,000 to 28,000 rpm from two thrombomodulin preparations, at protein concentrations from 0.01 to 0.07%, and in buffer containing 0.01 to 0.23% Lubrol. Examination of the data from different rotor speeds shows that the thrombomodulin exists as a heterogeneous mixture containing monomer (Mr 65,000), trimer, and higher oligomers. The oligomers do not equilibrate over the time scale of the experiment. The weight fraction as monomer varies from preparation to preparation, and appears to be independent of detergent concentration. Thus, experimenters should be cautious when interpreting binding or kinetic results obtained under similar buffer conditions.
