RESUMO
The semisynthetic vitamin E derivative alpha-tocopheryloxyacetic acid (α-TEA) induces tumor cell apoptosis and may offer a simple adjuvant supplement for cancer therapy if its mechanisms can be better understood. Here we report that α-TEA also triggers tumor cell autophagy and that it improves cross-presentation of tumor antigens to the immune system. α-TEA stimulated both apoptosis and autophagy in murine mammary and lung cancer cells and inhibition of caspase-dependent apoptosis enhanced α-TEA-induced autophagy. Cell exposure to α-TEA generated double-membrane-bound vesicles indicative of autophagosomes, which efficiently cross-primed antigen-specific CD8(+) T cells. Notably, vaccination with dendritic cells pulsed with α-TEA-generated autophagosomes reduced lung metastases and increased the survival of tumor-bearing mice. Taken together, our findings suggest that both autophagy and apoptosis signaling programs are activated during α-TEA-induced tumor cell killing. We suggest that the ability of α-TEA to stimulate autophagy and enhance cross-priming of CD8(+) T cells might be exploited as an adjuvant strategy to improve stimulation of antitumor immune responses.
Assuntos
Adenocarcinoma/tratamento farmacológico , Apresentação de Antígeno/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Mamárias Animais/tratamento farmacológico , Tocoferóis/farmacologia , Adenocarcinoma/imunologia , Adenocarcinoma de Pulmão , Animais , Apoptose/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células Dendríticas/imunologia , Feminino , Neoplasias Pulmonares/imunologia , Ativação Linfocitária , Neoplasias Mamárias Animais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , VacinaçãoRESUMO
Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal-regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development.
Assuntos
Apoptose/efeitos dos fármacos , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Adulto , Apoptose/genética , Astrocitoma/tratamento farmacológico , Astrocitoma/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Proliferação de Células/efeitos dos fármacos , Criança , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/administração & dosagem , Receptores Proteína Tirosina Quinases/genética , Células Tumorais Cultivadas , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Chemoresistance is a common cause of treatment failure in patients with acute myeloid leukemia (AML). We generated a diphtheria toxin (DT) fusion protein composed of the catalytic and translocation domains of DT (DT388) fused to interleukin-3 (IL-3). IL-3 receptors (IL-3R) are overexpressed on blasts from many AML patients. DT388IL-3 showed cytotoxicity to leukemic blasts in vitro and in vivo and minimal damage to normal tissues in nonhuman primate models. However, only a fraction of patient leukemic samples were sensitive to the agent. To enhance the potency and specificity of the DT388IL-3 molecule, we constructed variants with altered residues in the IL-3 moiety. Two of these variants, DT388IL-3[K116W] and DT388IL-3[Delta125-133], were produced and partially purified from Escherichia coli with excellent yields. They showed enhanced binding to the human IL-3R and greater cytotoxicity to human leukemia cell lines relative to wild-type DT388IL-3. Interestingly, the results support a previously hypothesized model for interaction of the C-terminal residues of IL-3 with a hydrophobic patch on the alpha-subunit of IL-3R. Rational modification of the targeting domain based on structural analysis can produce a fusion toxin with increased ability to kill tumor cells. One or both of these variant fusion proteins merit further development for therapy of chemotherapy refractory AML.