RESUMO
In this study, neural stem cells (NSCs)-derived enzyme/prodrug therapy (NDEPT) was used to treat primary lung cancer or metastatic lung cancer in the brain. To confirm the anti-tumor effect of NSCs expressing carboxyl esterase (CE), A549 lung cancer cells were treated with HB1.F3.CE cells and CPT-11. A significant decrease in the viability/proliferation of lung cancer cells was observed compared to negative controls or cells treated with CPT-11 alone. To produce a mouse model of primary lung cancer or lung cancer metastasis to the brain, A549 cells were implanted in the dorsal area of the mouse or right hemisphere. CM-DiI pre-stained stem cells were implanted near the primary lung cancer tumor mass or in the contralateral brain. Two days after stem cells injection, mice were inoculated with CPT-11 (13.5 kg/mouse/day) via intraperitoneal injection. In the primary lung cancer mouse models, tumor mass was 80% lower in response to HB1.F3.CE in conjunction with CPT-11, while it was only reduced by 40% in the group treated with CPT-11 alone. Additionally, therapeutic efficacy of co-treatment with stem cells and CPT-11 was confirmed by detection of apoptosis and necrosis in primary and metastatic lung cancer tissues. By secreting VEGF, tumor cells modulate Erk1/2 and Akt signaling and migration of stem cells. This further increased tumor-selectivity of stem cell/prodrug co-therapy. Overall, these results indicate that NSCs expressing the therapeutic gene may be a powerful tool for treatment of primary lung cancer or metastasis of lung cancer to the brain.
Assuntos
Camptotecina/análogos & derivados , Carboxilesterase/biossíntese , Neoplasias Pulmonares/terapia , Células-Tronco Neurais/transplante , Animais , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Carboxilesterase/genética , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Irinotecano , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Células-Tronco Neurais/enzimologia , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs). METHODS: NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-ßIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed. RESULTS: ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs. CONCLUSION: ICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Animais , Agregação Celular/efeitos dos fármacos , Agregação Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Células-Tronco Neurais/enzimologiaRESUMO
Strokes are devastating as there are no current therapies to prevent the long term neurological deficits that they cause. Soon after ischemic stroke, there is proliferation and differentiation of neural stem/progenitor cells as an important mechanism for neuronal restoration. However, endogenous neurogenesis by itself is insufficient for effective brain repair after stroke as most newborn neurons do not survive. One fascinating strategy for stroke treatment would thus be maintaining the survival and/or promoting the differentiation of endogenous neural stem/progenitor cells. Using transgenic (Tg) mice over-expressing the C. elegans fat-1 gene encoding an enzyme that converts endogenous omega-6 to omega-3 polyunsaturated fatty acids (n-3 PUFAs), we showed that fat-1 Tg mice with chronically elevated brain levels of n-3 PUFAs exhibited less brain damage and significantly improved long-term neurological performance compared to wild type littermates. Importantly, post-stroke neurogenesis occurred more robustly in fat-1 Tg mice after focal ischemia. This was manifested by enhanced neural stem cell proliferation/differentiation and increased migration of neuroblasts to the ischemic sites where neuroblasts matured into resident neurons. Moreover, these neurogenic effects were accompanied by significantly increased oligodendrogenesis. Our results suggest that n-3 PUFA supplementation is a potential neurogenic and oligodendrogenic treatment to naturally improve post-stroke brain repair and long-term functional recovery.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/genética , Ácidos Graxos Dessaturases/biossíntese , Ácidos Graxos Ômega-3/biossíntese , Neurogênese , Fármacos Neuroprotetores/metabolismo , Acidente Vascular Cerebral/enzimologia , Animais , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular/genética , Proliferação de Células , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/genética , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Transgenes/genéticaRESUMO
Intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration induces apoptosis of subventricular zone (SVZ) doublecortin (Dcx)-positive neural progenitor cells (migrating neuroblasts, A cells). Actually, a metabolite of MPTP, 1-methy-4-phenylpiridinium (MPP(+)), is responsible for neural progenitor cell toxicity. In the present study, to examine whether the MPTP-induced SVZ cell apoptosis is caused directly by MPP(+) metabolized through monoamine oxidase B (MAO-B), MPTP or MPP(+) was intracerebroventricularly (icv) injected into C57BL/6 mice. At Day 1 postinjection, many terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were observed in the SVZ of both low (36 µg) and high (162 µg) dose MPTP- and MPP(+)-injected mice. The number of Dcx-positive A cells showed a significant decrease following high dose of MPTP- or MPP(+)-injection on Days 1 and 3, respectively, whereas that of EGFR-positive C cells showed no change in mice with any treatment. In addition, prior icv injection of a MAO-B inhibitor, R(-)-deprenyl (deprenyl), inhibited MPTP-induced apoptosis, but not MPP(+)-induced apoptosis. MAO-B- and GFAP-double positive cells were detected in the ependyma and SVZ in all mice. It is revealed from these results that icv injection of MPTP induces apoptosis of neural progenitor cells (A cells) in the SVZ via MPP(+) toxicity. In addition, it is suggested that the conversion from MPTP to MPP(+) is caused mainly by MAO-B located in ependymal cells and GFAP-positive cells in the SVZ.