RESUMO
Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.
Assuntos
Neoplasias da Mama/terapia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Fotoquimioterapia , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/uso terapêutico , Ácido Aminolevulínico/toxicidade , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Metástase Neoplásica , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Survivina , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
A new water-soluble phthalocyanine derivative, 2,3,9,10,16,17,23,24-octakis(3-aminopropyloxy) phthalocyaninato zinc II (PoII) was studied as a photosensitizer for photodynamic therapy (PDT) in MCF-7c3 cells. We report here that PoII and red light induces apoptosis. However, the precise mechanism appears to differ from that induced by PDT with other known phthalocyanines. The present study provides evidence that in the case of PoII, caspases do not participate in the apoptotic response. PoII-PDT-treated cells exhibited chromatin condensation and phosphatidylserine (PS) externalization. In the absence of light activation, PoII had no detectable cytotoxic effect. An early event upon PoII-PDT was photodamage to lysosomes, suggesting that they are the primary sites of action. Moreover, the treatment induces Bid activation, mitochondrial swelling and translocation of apoptosis-inducing factor (AIF) to the nucleus. An atypical proteolysis of poly(ADP-ribose) polymerase (PARP) indicative of calpain-like activation was observed. These data support the notion that an alternative mechanism of caspase-independent apoptosis was found in PoII-photosensitized cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Indóis/farmacologia , Indóis/uso terapêutico , Compostos Organometálicos/farmacologia , Compostos Organometálicos/uso terapêutico , Fotoquimioterapia , Água/química , Fator de Indução de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neoplasias da Mama/enzimologia , Calpaína/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Fase G1/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Transporte Proteico/efeitos dos fármacos , Solubilidade/efeitos dos fármacosRESUMO
Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.
Assuntos
Apoptose , Neoplasias da Mama/patologia , Inativação Gênica/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Caspases , Feminino , Humanos , Proteínas Inibidoras de Apoptose , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Survivina , TransfecçãoRESUMO
The aim of the present study was to evaluate the photodynamic efficacy of a novel phthalocyanine derivate 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino) ethylsulfanyl]phthalocyaninatozinc(II) (referred here as S1) using MCF-7c3 human breast cancer cells and the LM2 adenocarcinoma subcutaneously implanted in Balb/c mice as experimental models. The S1-l-alpha-dimyristoyl-phosphatidylcholine liposome was selected as the best delivery system because it showed greater internalization into cells (35 nmol/10(6) cells), relative to other liposomes. After 3 h incubation S1 was partially localized in lysosomes, the compartment that represented its primary photodamage site. The S1 treated cultures also revealed a degree of mitochondrial morphology alteration. Indeed, S1 leads to photokilling of the cells with different efficacies indicating that cell photoinactivation was dependent on both the phthalocyanine concentration and the light dose applied. Analyses of morphology and nuclear condensation level indicated that some of the cells exposed to photodynamic therapy were undergoing apoptosis within 8h after treatment. To assess the in vivo effectiveness of S1, animals bearing tumors were treated with 0.2mg/kg S1 followed 24h later by 108 J cm(-2) light at 600-800 nm and 60 mW cm(-2),while other animals served as controls (no treatment, light alone, or S1 alone). All S1 treated tumors and none of the controls exhibited complete or partial responses, and these responses continued for the entire observation period of 12 days. Evaluation of tumor size showed that the treatment effectively delayed tumor growth. Light microscopy investigations of irradiated tumor specimens showed that S1 causes an early direct damage of malignant cells, largely via processes leading to random necrotic pathways.