RESUMO
Paeonol is a major phenolic micromolecular component of Moutan cortex Radicis, a traditional Chinese Medicine. It has shown antitumor effects in previous studies; however, the underlying mechanisms remain unknown. This study investigated the mechanism by giving treatments of placebo, cyclophosphamide, paeonol of 150 and 300 mg/kg to 4 groups of mice bearing EMT6 breast cancer. Apoptosis in tumor cells were confirmed by morphology analysis, including hematoxylin, eosin staining and TUNEL staining. The results showed that the weight of EMT6 breast tumor was significantly reduced in the groups treated with both 150 and 300 mg/kg of paeonol. Immunohistochemical and Western blot results showed that the expression of Bcl-2 was down-regulated while the expression of Bax, caspase 8 and caspase 3 was up-regulated respectively. These results suggest that paeonol exhibits antitumor effects and the mechanism of the inhibition is via induction of apoptosis, regulation of Bcl-2 and Bax expression, and activation of caspase 8 and caspase 3.
RESUMO
OBJECTIVES: Human lactoferrin, an 80 kDa iron-binding glycoprotein, has antitumour effects. We have explored the potential therapeutic role of re-expressing human lactoferrin gene product in human breast cancer. METHODS: A recombinant adenovirus expressing the human lactoferrin cDNA (ad-hLTF) was constructed and used to infect breast cancer cells. KEY FINDINGS: Seventy-two hours after infection, ad-hLTF had considerable cytotoxicity on MCF-7 cells. A time-course study showed that ad-hLTF infection of MCF-7 cells at 100 plaque-forming units per cell increased the number of cells in G(0) /G(1) phase and appeared markedly at Sub-G(1) apoptotic peak. The presence of apoptotic cells was confirmed using Annexin V-fluoresecein isothiocyanate apoptosis detection by flow cytometry. Ad-hLTF also resulted in a decrease of Bcl-2 protein and an increase in Bax protein. CONCLUSIONS: Ad-hLTF plays an important role in the induction of cell cycle arrest and apoptosis in MCF-7 cells. The results demonstrated that ad-hLTF could have potential benefits in the treatment of breast cancer.
Assuntos
Adenoviridae/genética , Apoptose/efeitos dos fármacos , Terapia Genética , Lactoferrina/genética , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Neoplasias da Mama/patologia , Caspase 3/genética , Ciclo Celular/genética , DNA Complementar/genética , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genética , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/genéticaRESUMO
Human lactoferrin (hLTF) is an 80KD iron-binding protein. It has been reported that hLTF exists anti-tumor effects. In this study Adenovirus Vectors Mediated Human Lactoferrin cDNA (ad-rhLTF) was constructed and an antitumor effects of ad-rhLTF were investigated in mice bearing EMT6 breast carcinoma. The results demonstrated that ad-rhLTF (5 x 10(8) and 25 x 10(8) pfu/ml local injection) had high expression in tumor tissues and effectively reduced the weight of EMT6 breast tumors. Compared with the control group, cell cycle assay by flow cytometry showed that ad-rhLTF increased the percentage of tumor cells in the Sub-G1 phase and G0/G1 phase and the apoptotic number reached to 23.2% in ad-rhLTF group (25 x 10(8) pfu/ml). Ad-rhLTF treatment also resulted in a decrease of Bcl-2 and an increase in Bax and caspase 3 expressions, which was demonstrated by immunohistochemical analysis and RT-PCR. These data suggest that the antitumor effects of ad-rhLTF might be associated with arresting tumor cells in the G0/G1 phase, inducing cell apoptosis and regulation of the expression of Bcl-2, Bax and activation of caspase 3.
Assuntos
Adenoviridae , Neoplasias da Mama/terapia , DNA Complementar/genética , Vetores Genéticos , Lactoferrina/genética , Animais , Western Blotting , Caspase 3/biossíntese , Caspase 3/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , DNA Complementar/biossíntese , Feminino , Citometria de Fluxo , Terapia Genética , Proteínas de Fluorescência Verde , Humanos , Lactoferrina/biossíntese , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Human lactoferrin (hLTF), an 80-kDa iron-binding glycoprotein, has antitumor activity. In this study, a recombinant adenovirus containing the human lactoferrin cDNA (ad-rhLTF) was constructed and its effect on tumor growth was investigated in mice bearing EMT6 breast cancer. Ad-rhLTF was injected seven times within 14 days into the tumor site at two concentrations (10(8) and 5 × 10(8) pfu/mL) in mice bearing EMT6 breast cancer. Injected ad-rhLTF had considerable cytotoxicity on mice breast cancer, and significantly reducing the weight of tumor produced and increasing the tumor inhibition rate up to 52.64%. The presence of apoptotic cells was confirmed using TUNEL staining and flow cytometry assays. At the same time, RTPCR and Western blot analyses demonstrated that ad-rhLTF also decreased expression of Bcl-2 and increased Bax and caspase 3 expressions. Therefore, we conclude that ad-rhLTF inhibits tumor growth by inducing tumor cell apoptosis in mice with breast cancer by triggering the mitochondrial-dependent pathway and activation of caspase 3. The results indicate that ad-rhLTF might be a promising drug for breast cancer gene therapy.