ABSTRACT
OBJECTIVE@#To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism.@*METHODS@#Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels, self-renewal capability (Nanog and Sox2), and cancer stem cell markers (CD133 and CD44), were detected by a colorimetric DNMT activity/inhibition assay kit, quantitative real-time reverse transcription-polymerase chain reaction, sphere and colony formation assays, and immunoblot, respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA, respectively, were performed to explore the mechanism for action of CAS (0, 10, 30, and 100 nmol/L).@*RESULTS@#DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (P<0.05). In addition, HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities, and lower CD133, CD44, Nanog and Sox2 protein expressions (P<0.05). Conversely, overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore, CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (P<0.05). Interestingly, DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected, DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells.@*CONCLUSION@#CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation, suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.
Subject(s)
Female , Humans , Cell Line, Tumor , HeLa Cells , Neoplastic Stem Cells/metabolism , RNA, Small Interfering/metabolism , Uterine Cervical Neoplasms/metabolismABSTRACT
To investigate the mechanism of inhibitory effect of a novel bFGF antagonist peptide isolated from the phage display random heptapeptide library on cell proliferation induced by basic fibroblast growth factor. The effect of P7 on cell morphology was observed under an inverted microscope. Flow cytometry was applied to analyze the effect of P7 on cell cycle progress of bFGF-stimulated cells. The effect of P7 on bFGF-induced activation of MEK and Erk1/2 in MAPK pathway was detected by Western blotting. The results showed that no significant cell morphology change was observed in the range of detected concentrations of P7. Cell cycle analysis showed that P7 decreased S-phase cell population and arrested cell cycle at the G0/G1 phase of bFGF-stimulated cells. The results of MAP kinase activation assay indicated that P7 decreased bFGF-induced MEK and Erk1/2 phosphorylation in a dose-dependent manner. P7 inhibited proliferation of bFGF-stimulated Balb/c 3T3 cells possibly via cell cycle arrest at the G0/G1 phase and down-regulation of signal molecular activation in MAPK pathway.
Subject(s)
Animals , Mice , BALB 3T3 Cells , Cell Cycle , Cell Proliferation , Fibroblast Growth Factor 2 , Pharmacology , MAP Kinase Kinase Kinases , Metabolism , MAP Kinase Signaling System , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Peptides , Pharmacology , Phosphorylation , Protein BindingABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of cell surface sialic acid and its linkage on the cell-cell and cell-matrix adhesion of mammary carcinoma cells MD-MB-435.</p><p><b>METHODS</b>MD-MB-435 cells were sense-transfected with ST6Gal I cDNA or antisense-transfected with part of the ST6Gal I sequence inserted in pcDNA 3.1 vector, with mock transfection with pcDNA3.1 vector as the control. The cell surface alpha2, 6-linked sialylation was determined by fluorescence-activated cell sorting (FACS) using lectin SNA (Sambucus nigra agglutinin specific to alpha2, 6-linked sialic acid on N-linked glycoprotein). A significantly increased alpha2, 6-sialylation subclone in sense-transfectants and a decreased alpha2, 6-sialylation subclone in antisense-transfectants were selected for further examination of cell-cell and cell-matrix (collagen IV) adhesion. The transfectants were also treated with sialidase to compare the capacity of cell adhesion affected by cell surface sialylation.</p><p><b>RESULTS</b>Sense-transfection subclone showed a reduced cell-cell aggregation but enhanced cell-matrix adhesion. In contrast, the antisense-transfection subclone exhibited increased cell-cell aggregation and decreased cell-matrix adhesion. After treatment with sialidase, the cell-matrix adhesion of all the transfectants and the parental MDA-MB-435 cells were significantly reduced to the level of 31%-57% of untreated cells.</p><p><b>CONCLUSION</b>Cell surface sialic acid and alpha2, 6-linked sialylation play an important role in cell-cell and cell-matrix adhesion of mammary carcinoma cell MDA-MB-435.</p>