RESUMO
NS-9 is a complex of polyinosinic-polycytidylic acid and a novel cationic liposome, LIC-101. The complex has strong cytotoxic activity against tumor cells derived from epithelial or fibroblastic cells. We have investigated the mechanism of the cytotoxic activity of NS-9 using knockdown cells in which the expression of proteins of interest was inhibited by RNA interference. NS-9 showed strong cytotoxic activity against knockdown cells with reduced expression of double-stranded RNA-dependent protein kinase, RNase L, or IFN-alpha/beta receptor, but showed no cytotoxic activity against IFN regulatory factor-3 (IRF3) knockdown cells. In IRF3-knockdown cells, NS-9 also did not induce either the DNA fragmentation or the rRNA degradation observed in negative control cells. We conclude that IRF3 plays a crucial role in the cytotoxic activity of NS-9 against tumor cells, whereas RNA-dependent protein kinase, RNase L, or type I IFNs are not important for its activity.
Assuntos
Apoptose , Cátions , Proteínas de Ligação a DNA/metabolismo , Lipossomos , Poli I-C/farmacologia , Interferência de RNA , Fatores de Transcrição/metabolismo , Antineoplásicos/antagonistas & inibidores , Antineoplásicos/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Células HeLa/efeitos dos fármacos , Humanos , Fator Regulador 3 de Interferon , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/antagonistas & inibidores , Interferon beta/genética , Interferon beta/metabolismo , RNA Ribossômico/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
PURPOSE: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel cationic liposome, LIC-101. EXPERIMENTAL DESIGN: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. RESULTS: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. CONCLUSIONS: By combining siRNA with our cationic liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.
Assuntos
Antineoplásicos/farmacologia , Cátions/química , Lipossomos/química , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/química , Animais , Western Blotting , Linhagem Celular Tumoral , DNA/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias/genética , Oligonucleotídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , RNA Interferente Pequeno/metabolismo , Fatores de TempoRESUMO
The pharmacokinetics and antitumor activity of pegylated small interfering RNA (siRNA)/cationic liposome complexes were studied after systemic administration to mice. We designed pegylated-lipid carriers for achieving increased plasma concentrations of RNA and hence improved accumulation of RNA in tumors by the enhanced permeability and retention effect. We compared the pharmacokinetics of siRNA complexed with liposomes incorporating pegylated lipids with longer (C-17 or C-18), shorter (C-12 to C-16), or unsaturated (C-18:1) acyl chains. When longer acyl chains were used, the plasma concentrations of siRNA obtained were dramatically higher than when shorter or unsaturated chains were used. This may be explained by the higher gel-to-liquid-crystalline phase-transition temperature (Tc) of lipids with longer acyl chains, which may form more rigid liposomes with reduced uptake by the liver. We tested a siRNA that is sequence specific for the antiapoptotic bcl-2 mRNA complexed with a pegylated liposome incorporating a C-18 lipid (PEG-LIC) by i.v. administration in a mouse model of human prostate cancer. Three-fold higher accumulation of RNA in the tumors was achieved when PEG-LIC rather than nonpegylated liposomes was used, and sequence-specific antitumor activity was observed. Our siRNA/PEG-LIC complex showed no side effects on repeated administration and the strength of its antitumor activity may be attributed to its high uptake by the tumors. Pegylation of liposomes improved the plasma retention, uptake by s.c. tumors, and antitumor activity of the encapsulated siRNA. PEG-LIC is a promising candidate for siRNA cancer therapy.