RESUMEN
The cancer stem cell hypothesis suggests that rare populations of tumor-initiating cells may be resistant to therapy, lead to tumor relapse and contribute to poor prognosis for cancer patients. We previously demonstrated the feasibility of p53 pathway restoration in p53-deficient tumor cell populations using small molecules including ellipticine or its derivatives. We now establish a single cell p53-regulated green fluorescent protein (EGFP)-reporter system in human DLD1 colon tumor cells expressing mutant p53 protein. We use these p53-EGFP reporter DLD1 cells to investigate the status of p53 transcriptional activity in putative colon cancer stem cell populations following exposure to p53 pathway-restoring drugs and/or classical chemotherapy. We demonstrate induction of p53-specific EGFP reporter fluorescence following overexpression of p53 family member p73 by an Adenovirus vector. We further show that p53-reporter activity is induced in DLD1 putative cancer stem cell side-populations analyzed by their Hoechst dye efflux properties following treatment with the p53 pathway restoring drug ellipticine. Combination of ellipticine with the cytotoxic agent 5-fluorouracil resulted in increased cytotoxicity as compared to either agent alone and this was associated with depletion of putative cancer stem cell populations as compared with 5-FU alone treatment. Our results support the feasibility of therapeutic targeting of mutant p53 in putative cancer stem cells as well as the potential to enhance cytotoxic chemotherapy.
Asunto(s)
Adenocarcinoma/patología , Antineoplásicos/farmacología , Neoplasias del Colon/patología , Elipticinas/farmacología , Fluorouracilo/farmacología , Genes p53 , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/efectos de los fármacos , Antineoplásicos/administración & dosificación , Proteínas de Unión al ADN/fisiología , Sinergismo Farmacológico , Elipticinas/administración & dosificación , Fluorouracilo/administración & dosificación , Genes Reporteros , Genes Sintéticos , Vectores Genéticos/farmacología , Humanos , Mutación , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/fisiología , Pirimidinas/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Proteínas Recombinantes de Fusión/fisiología , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
The cancer stem cell theory describes a small subset of cancer cells that have the ability to initiate and drive the growth of a tumor. The niche refers to the environmental factors and the surrounding cells within which the tumor develops. The exact relationship between cancer stem cells and the tumor niche is not known. However, using side population analysis by flow cytometry, it is possible to analyze the relationship between environmental stresses and putative cancer stem cells. The side population is a subpopulation of cells that efflux Hoechst 33342 and has been previously shown to be enriched for cancer stem cells. Using this technique, we characterized the response of side population cells to low confluency, serum starvation and hypoxia using three different human cancer cell lines. We found that these stresses, characteristic of the tumor niche enrich the side population of DLD1, SW480 and MCF7 cancer cell lines, thus possibly predisposing the tumor to a more malignant phenotype.
