RESUMEN
BACKGROUND: Cryptochrome 1 (CRY1) is a key protein that regulates the feedback loop of circadian clock. The abnormal expression of CRY1 was reported in numerous cancers, and contributed to tumorigenesis and progression. But the underlying mechanism remains undefined. METHODS: CRY1 overexpression was constructed by lentivirus vector. Gene and protein expression was detected by reverse transcription quantitative polymerase chain reaction and Western blot. Cell proliferation was analyzed by CCK-8 assay. Cell migration ability was analyzed by scratch assay and transwell migration assay. The cAMP concentration was measured by intracellular cAMP assay. RESULTS: Overexpression of CRY1 showed slightly effect on the proliferation and migration of HGC-27 cells. Upon exposure to isoproterenol (ISO), a ß-adrenergic receptor agonist, cell proliferation, and migration were inhibited while the cAMP/PKA pathway was activated and ERK1/2 phosphorylation was suppressed. CRY1 overexpression reduced cAMP accumulation, retained ERK1/2 phosphorylation level and alleviated the antiproliferative effect upon exposure to ISO. However, CRY1 overexpression was inoperative on the antiproliferative effect of forskolin (FSK), a direct activator of adenyl cyclase (AC), or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase (PDE) inhibitor. CONCLUSIONS: Our results suggest CRY1 overexpression may protect cells from the antiproliferative effects via activation of the cAMP/PKA pathway through interrupting signal transduction from G protein-coupled receptors to AC.
RESUMEN
The partitioning of nucleic acids is sensitive to pH during phenol extraction. However, the exact effects of pH on phenol extraction had not been systematically investigated, and the mechanism of which were not fully elucidated. In this paper, we showed that the partitioning of nucleic acids was determined neither solely by the pH of the aqueous buffer being used, nor by the "pH of the phenol"; the latter is a completely wrong conception. We demonstrated that a key determinant for nucleic acid partitioning during phenol extraction was the equilibrated pH of the aqueous phase, which should be defined as the pH of phenol extraction. For example, when 50 mM NaAc-HAc buffer at pH of 3.47 was mixed with an equal volume of water-saturated phenol, the equilibrated pH of aqueous phase would be raised to â¼3.84. At this pH, almost all of genomic DNA partitioned into the phenol phase, and genomic DNA-free total RNA was retained in the aqueous phase. Several salts were found affecting the partitioning of nucleic acids during phenol extraction in different manners. Based on these results, a low-cost and efficient method for genomic DNA-free total RNA extraction was developed.
Asunto(s)
Extractos Celulares/química , Fenol/química , ARN/química , Escherichia coli , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Extracción Líquido-Líquido/métodos , Sales (Química)/química , Solventes/químicaRESUMEN
BACKGROUND TCL-based immunotherapy has been applied in the field cancer therapy. However, it is un clear whether this therapy can be used to treat triple-negative breast cancer (TNBC), and different TNBC cells have distinct responses to this therapy. MATERIAL AND METHODS In the present work, we conducted 2 different TCL-based immunotherapies to treat TNBC and compared their anti-tumor effect on 4 TNBC cell lines: MDA-MB-231, MDA-MB-436, HCC1937, and HCC1187. RESULTS Peripheral blood mononuclear cells (PBMC) activated by TCL and peripheral blood lymphocytes (PBL) stimulated with TCL-loaded DC demonstrated the ability to kill TNBC cells in vitro, but the killing efficiency of PBL was much higher than that of PBMC. In vivo, PBL stimulated with TCL-loaded DC can also stop the growth of TNBC tumors in mice. HCC1187 and MDA-MD-231 best respond to TCL-based immunotherapy both in vitro and in vivo. The response of HCC1937 was weaker, and that of MDA-MB-436 was lowest among the 4 cell lines. Total mRNA microarray analysis of TNBC cells showed that PDL-1 mRNA expression in HCC1937 and MDA-MD-436 cells was higher than in the other 2 TNBC cell lines, and that of MDA-MB-436 was higher than that of HCC1937. PD1 blocking can decrease the apoptosis rate. These results show that different contents of PDL-1 in TCL, by interacting with PD expression on lymphocytes, can induce different ratios of lymphocyte apoptosis, and then result in distinct response of the 4 TNBC cell lines to TCL-based immunotherapy. CONCLUSIONS TCL-based immunotherapy has discrepant anti-tumor efficiency in different TNBC cell lines by PDL-1/PD interaction, providing the theoretical basis of TCL-based immunotherapy in TNBC.