RESUMO
OBJECTIVE: To study on the cultivation method for tumor spheres from colorectal cancer cell lines and identify whether resulting Colo205 spheroid cells display cancer stem cell characteristics. METHODS: Lovo, Colo205 and SW480 cells were seeded in serum free medium (SFM) with EGF and bFGF. Flow cytometry analysis, cell invasion assay and xenograft experiment were applied to examine the cell surface marker expression pattern, cell invasive ability and in vivo tumorigenicity of both Colo205 spheres and parental cells. CD44 expression of tumor spheroid cells was also analyzed after cultured with serum supplemented medium by flow cytometry. CD44, Musashi-1 and Oct4 mRNA were detected in these two cells by RT-PCR. RESULTS: Tumor spheres could be generated from three colorectal cancer cell lines in SFM. The formation and proliferation of tumor spheres were benefited from fresh SFM, cell dissociation reagent Accutase and the floating status of cancer cells. The overwhelming majority of spheroid cells were CD44+ cells. But CD44+ cells were gradually decreased when spheres cultured with serum supplemented medium. Colo205 spheres have higher Musashi-1 and Oct4 mRNA expression, tumor-initiating capability and invasive ability compared with those of parental cells. CONCLUSION: Tumor spheres in which enrich cancer stem cells can be generated and matained from colorectal cancer cell lines in SFM on floating-culture condition.
Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/patologia , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/citologia , Esferoides Celulares/citologia , Linhagem Celular Tumoral , Humanos , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Esferoides Celulares/metabolismoRESUMO
OBJECTIVE: To explore the effect of Rac1 siRNA on the expression of Rac1 and the biological behaviors of gastrointestinal cancer cells. METHODS: Rac1 siRNA was transfected into human gastric cancer cell line SGC803 and colorectal cancer cell line Lovo by lipofectamine 2000. The expression of Rac1 in these cell lines were detected by Western blot and RT-PCR after 48 hours of the transfection. The effect of Rac1 on the proliferation of human gastric cancer cell line SGC803 and colorectal cancer cell line Lovo were tested by CCK8 kit. The motility of the transfected and the control cancer cells were assessed by Wound-healing assay and invasion assay. The apoptotic index was evaluated by Hoechst 33258 staining and FCM. RESULTS: Rac1 siRNA can down-regulated the expression of Rac1 on human gastric cancer cell line SGC803 and colorectal cancer cell line Lovo remarkably, and Rac1 siRNA can inhibit both the proliferation and motility of the transfectants. Analysis of apoptosis demonstrated that Rac1 siRNA can promote apoptosis of the gastric cancer cells and colorectal cancer cells. CONCLUSION: Rac1 play an important role in the regulation of biological behaviors of human gastric cancer and colorectal cancer cells, and the interference of Rac1 expression could provide a novel path in reversing the malignant phenotypes of these malignancies.